Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 1.4-butanediol related thereto

ABSTRACT

Provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway (MMP) that can enhance the availability of reducing equivalents in the presence of methanol. Such reducing equivalents can be used to increase the product yield of organic compounds produced by the microbial organism, such as 1,4-butanediol (BDO). Also provided herein are methods for using such an organism to produce BDO.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/424,404, filed Feb. 26, 2015, which is a 371 national stageapplication of international application Serial No. PCT/US2013/056725filed Aug. 27, 2013, which is a continuation-in-part of U.S. Ser. No.13/975,678 filed Aug. 26, 2013, and claims the benefit of U.S. Ser. No.61/766,609 filed Feb. 19, 2013, and U.S. Ser. No. 61/693,683 filed Aug.27, 2012, each of which is incorporated herein by reference in itsentirety.

1. SUMMARY

Provided herein are methods generally relating to metabolic andbiosynthetic processes and microbial organisms capable of producingorganic compounds. Specifically, provided herein is a non-naturallyoccurring microbial organism (NNOMO) having a methanol metabolic pathway(MMP) that can enhance the availability of reducing equivalents in thepresence of methanol and/or convert methanol to formaldehyde. Such NNOMOand reducing equivalents can be used to increase the product yield oforganic compounds produced by the microbial organism, such as1,4-butanediol (BDO). Also provided herein are NNOMOs and methodsthereof to produce optimal yields of BDO.

In a first aspect, provided herein is a NNOMO having a methanolmetabolic pathway (MMP), wherein said organism comprises at least oneexogenous nucleic acid encoding a MMP enzyme (MMPE) expressed in asufficient amount to enhance the availability of reducing equivalents inthe presence of methanol. In certain embodiments, the MMP comprises oneor more enzymes selected from the group consisting of a methanolmethyltransferase (EM1); a methylenetetrahydrofolate reductase (EM2); amethylenetetrahydrofolate dehydrogenase (EM3); amethenyltetrahydrofolate cyclohydrolase (EM4); a formyltetrahydrofolatedeformylase (EM5); a formyltetrahydrofolate synthetase (EM6); a formatehydrogen lyase (EM15); a hydrogenase (EM16); a formate dehydrogenase(EM8); a methanol dehydrogenase (EM9); a formaldehyde activating enzyme(EM10); a formaldehyde dehydrogenase (EM11); aS-(hydroxymethyl)glutathione synthase (EM12); a glutathione-dependentformaldehyde dehydrogenase (EM13); and an S-formylglutathione hydrolase(EM14). Such organisms advantageously allow for the production ofreducing equivalents, which can then be used by the organism for theproduction of BDO using any one of the BDO pathways (BDOPs) providedherein.

In one embodiment, the MMP comprises an EM9. In another embodiment, theMMP comprises an EM9 and an EM10. In other embodiments, the MMPcomprises an EM1 and an EM2. In one embodiment, the MMP comprises anEM9, an EM3, an EM4 and an EM5. In another embodiment, the MMP comprisesan EM9, an EM3, an EM4 and an EM6. In other embodiments, the MMPcomprises an EM9 and an EM11. In another embodiment, the MMP comprisesan EM9, a EM12, and EM13 and an EM14. In other embodiments, the MMPcomprises an EM9, an EM13 and an EM14. In an embodiment, the MMPcomprises an EM9, an EM10, an EM3, an EM4 and an EM5. In anotherembodiment, the MMP comprises an EM9, an EM10, an EM3, an EM4 and anEM6. In other embodiments, the MMP comprises an EM1, an EM2, an EM3, andEM4, and EM5. In one embodiment, the MMP comprises an EM1, an EM2, anEM3, an EM4 and EM6. In certain of the above embodiments, the MMPfurther comprises an EM8. In other of the above embodiments, the MMPfurther comprises and EM15. In yet other of the above embodiments, theMMP further comprises an EM16. In certain embodiments, the organismcomprises two, three, four, five, six or seven exogenous nucleic acids,each encoding a MMPE.

In certain embodiments, the organism further comprises a 1,4-BDO pathway(BDOP). In certain embodiments, said organism comprises at least oneexogenous nucleic acid encoding a BDOPE expressed in a sufficient amountto produce BDO. In certain embodiments, the BDOPE is selected from thegroup consisting of a succinyl-CoA transferase (EB1) or a succinyl-CoAsynthetase (EB2A) (or succinyl-CoA ligase); a succinyl-CoA reductase(aldehyde forming) (EB3); a 4-hydroxybutyrate (4-HB) dehydrogenase(EB4); a 4-HB kinase (EB5); a phosphotrans-4-hydroxybutyrylase (EB6); a4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7); a1,4-butanediol dehydrogenase (EB8); a succinate reductase (EB9); asuccinyl-CoA reductase (alcohol forming) (EB10); a 4-hydroxybutyryl-CoAtransferase (EB11) or a 4-hydroxybutyryl-CoA synthetase (EB12); a 4-HBreductase (EB13); a 4-hydroxybutyryl-phosphate reductase (EB14); and a4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15).

In one embodiment, the BDOP comprises an EB3, an EB4, an EB5, an EB6, anEB7, and an EB8. In one embodiment, the BDOP comprises an EB3, an EB4,an EB11 or an EB12, an EB7, and an EB8. In one embodiment, the BDOPcomprises an EB3, an EB4, an EB11 or an EB12, and an EB15. In oneembodiment, the BDOP comprises an EB3, an EB4, an EB5, an EB6, and anEB15. In one embodiment, the BDOP comprises an EB3, an EB4, an EB13, andan EB8. In one embodiment, the BDOP comprises an EB3, an EB4, an EB5, anEB14, and an EB8. In one embodiment, the BDOP comprises an EB10, an EB5,an EB6, an EB7, and an EB8. In one embodiment, the BDOP comprises anEB10, an EB5, an EB6, and an EB15. In one embodiment, the BDOP comprisesan EB10, an EB11 or an EB12, an EB7, and an EB8. In one embodiment, theBDOP comprises an EB10, an EB11 or an EB12, and an EB15. In oneembodiment, the BDOP comprises an EB10, an EB13, and an EB8. In oneembodiment, the BDOP comprises an EB10, an EB5, an EB14 and an EB8. Inone embodiment, the BDOP comprises an EB9, an EB4, an EB5, an EB6, anEB7, and an EB8. In one embodiment, the BDOP comprises an EB9, an EB4,an EB11 or an EB12, an EB7, and an EB8. In one embodiment, the BDOPcomprises an EB9, an EB4, an EB11 or an EB12, and an EB15. In oneembodiment, the BDOP comprises an EB9, an EB4, an EB5, an EB6, and anEB15. In one embodiment, the BDOP comprises an EB9, an EB4, an EB13, andan EB8. In one embodiment, the BDOP comprises an EB9, an EB4, an EB5, anEB14, and an EB8. In certain of the above embodiments, the BDOP furthercomprises an EB1. In other of the above-embodiments, the BDOP furthercomprises an EB2A. In some embodiments, the organism comprises four,five, six or seven exogenous nucleic acids, each encoding a BDOPE.

In other embodiments, the organism having a MMP, either alone or incombination with a BDOP, as provided herein, further comprises aformaldehyde assimilation pathway (FAP) that utilizes formaldehyde,e.g., obtained from the oxidation of methanol, in the formation ofintermediates of certain central metabolic pathways that can be used,for example, in the formation of biomass. In certain embodiments, theorganism further comprises a FAP, wherein said organism comprises atleast one exogenous nucleic acid encoding a formaldehyde assimilationpathway enzyme (FAPE) expressed in a sufficient amount to produce anintermediate of glycolysis and/or a metabolic pathway that can be usedin the formation of biomass. In one embodiment, the FAPE is expressed ina sufficient amount to produce an intermediate of glycolysis. In anotherembodiment, the FAPE is expressed in a sufficient amount to produce anintermediate of a metabolic pathway that can be used in the formation ofbiomass. In some of the embodiments, the FAP comprises ahexulose-6-phosphate (H6P) synthase (EF1), a 6-phospho-3-hexuloisomerase(EF2), a dihydroxyacetone (DHA) synthase (EF3) or a DHA kinase (EF4). Inone embodiment, the FAP comprises an EF1 and an EF2. In one embodiment,the intermediate is a H6P, a fructose-6-phosphate (F6P), or acombination thereof. In other embodiments, the FAP comprises an EF3 oran EF4. In one embodiment, the intermediate is a DHA, a DHA phosphate,or a combination thereof. In certain embodiments, the organism comprisestwo exogenous nucleic acids, each encoding a FAPE.

In certain embodiments, provided herein is a NNOMO having a MMP, whereinsaid organism comprises at least one exogenous nucleic acid encoding anEM9 expressed in a sufficient amount to enhance the availability ofreducing equivalents in the presence of methanol and/or expressed in asufficient amount to convert methanol to formaldehyde. In someembodiments, the organism comprises at least one exogenous nucleic acidencoding an EM9 expressed in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol. Inother embodiments, the organism comprises at least one exogenous nucleicacid encoding an EM9 expressed in a sufficient amount to convertmethanol to formaldehyde. In some embodiments, the microbial organismfurther comprises a FAP. In certain embodiments, the organism furthercomprises at least one exogenous nucleic acid encoding a FAPE expressedin a sufficient amount to produce an intermediate of glycolysis. Incertain embodiments, the FAPE is selected from the group consisting ofan EF1, an EF2, an EF3 and an EF4.

In certain embodiments, at least one exogenous nucleic acid is aheterologous nucleic acid. In some embodiments, the organism is in asubstantially anaerobic culture medium. In some embodiment, themicrobial organism is a species of bacteria, yeast, or fungus.

In some embodiments, the organism further comprises one or more genedisruptions, occurring in one or more endogenous genes encodingprotein(s) or enzyme(s) involved in native production of ethanol,glycerol, acetate, lactate, formate, CO₂, and/or amino acids by saidmicrobial organism, wherein said one or more gene disruptions conferincreased production of BDO in said microbial organism. In someembodiments, one or more endogenous enzymes involved in nativeproduction of ethanol, glycerol, acetate, lactate, formate, CO₂ and/oramino acids by the microbial organism, has attenuated enzyme activity orexpression levels. In certain embodiments, the organism comprises fromone to twenty-five gene disruptions. In other embodiments, the organismcomprises from one to twenty gene disruptions. In some embodiments, theorganism comprises from one to fifteen gene disruptions. In otherembodiments, the organism comprises from one to ten gene disruptions. Insome embodiments, the organism comprises from one to five genedisruptions. In certain embodiments, the organism comprises 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 or 25 gene disruptions or more.

In another aspect, provided herein is a method of producingformaldehyde, comprising culturing a NNOMO provided herein underconditions and for a sufficient period of time to produce formaldehyde.In certain embodiment, the NNOMO comprises an exogenous nucleic acidencoding an EM9. In certain embodiments, the formaldehyde is consumed toprovide a reducing equivalent. In other embodiments, the formaldehyde isconsumed to incorporate into BDO or another target product.

In another aspect, provided herein is a method of producing anintermediate of glycolysis and/or a metabolic pathway that can be usedin the formation of biomass, comprising culturing a NNOMO providedherein under conditions and for a sufficient period of time to producethe intermediate In certain embodiment, the NNOMO comprises an exogenousnucleic acid encoding an EM9. In certain embodiments, the formaldehydeis consumed to provide a reducing equivalent. In other embodiments, theformaldehyde is consumed to incorporate into BDO or another targetproduct.

In another aspect, provided herein is a method for producing BDO,comprising culturing any one of the NNOMOs comprising a MMP and an BDOPprovided herein under conditions and for a sufficient period of time toproduce BDO. In certain embodiments, the organism is cultured in asubstantially anaerobic culture medium.

2. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary metabolic pathways enabling the extraction ofreducing equivalents from methanol. The enzymatic transformations shownare carried out by the following enzymes: 1A) a methanolmethyltransferase (EM1), 1B) a methylenetetrahydrofolate reductase(EM2), 1C) a methylenetetrahydrofolate dehydrogenase (EM3), 1D) amethenyltetrahydrofolate cyclohydrolase (EM4), 1E) aformyltetrahydrofolate deformylase (EM5), 1F) a formyltetrahydrofolatesynthetase (EM6), 1G) a formate hydrogen lyase (EM15), 1H) a hydrogenase(EM16), 1I) a formate dehydrogenase (EM8), 1J) a methanol dehydrogenase(EM9), 1K) a formaldehyde activating enzyme (EM10), 1L) a formaldehydedehydrogenase (EM11), 1M) a S-(hydroxymethyl)glutathione synthase(EM12), 1N) a glutathione-dependent formaldehyde dehydrogenase (EM13),and 1O) a S-formylglutathione hydrolase (EM14). In certain embodiments,steps K and/or M are spontaneous.

FIG. 2 shows exemplary BDOPs, which can be used to increase BDO yieldsfrom carbohydrates when reducing equivalents produced by a MMP providedherein are available. BDO production is carried out by the followingenzymes: 2A) a succinyl-CoA transferase (EB1) or a succinyl-CoAsynthetase (EB2A), 2B) a succinyl-CoA reductase (aldehyde forming)(EB3), 2C) a 4-HB dehydrogenase (EB4), 2D) a 4-HB kinase (EB5), 2E) aphosphotrans-4-hydroxybutyrylase (EB6), 2F) a 4-hydroxybutyryl-CoAreductase (aldehyde forming) (EB7), 2G) a 1,4-butanediol dehydrogenase(EB8), 2H) a succinate reductase (EB9), 21) a succinyl-CoA reductase(alcohol forming) (EB10), 2J) a 4-hydroxybutyryl-CoA transferase (EB11)or 4-hydroxybutyryl-CoA synthetase (EB12), 2K) a 4-HB reductase (EB13),2L) a 4-hydroxybutyryl-phosphate reductase (EB14), and 2M) a4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15).

FIG. 3 shows an exemplary FAP. The enzymatic transformations are carriedout by the following enzymes: 3A) a H6P synthase (EF1), and 3B) a6-phospho-3-hexuloisomerase (EF2).

FIG. 4 shows an exemplary FAP. The enzymatic transformations are carriedout by the following enzymes: 4A) a DHA synthase (EF3), and 4B) a DHAkinase (EF4).

3. DETAILED DESCRIPTION 3.1 Definitions

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism provided herein isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a BDO or 4-HBbiosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, NNOMOs can havegenetic modifications to nucleic acids encoding metabolic polypeptides,or functional fragments thereof. Exemplary metabolic modifications aredisclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” are intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

As used herein, the term “gene disruption,” or grammatical equivalentsthereof, is intended to mean a genetic alteration that renders theencoded gene product inactive or attenuated. The genetic alteration canbe, for example, deletion of the entire gene, deletion of a regulatorysequence required for transcription or translation, deletion of aportion of the gene which results in a truncated gene product, or by anyof various mutation strategies that inactivate or attenuate the encodedgene product. One particularly useful method of gene disruption iscomplete gene deletion because it reduces or eliminates the occurrenceof genetic reversions in the NNOMOs provided herein. The phenotypiceffect of a gene disruption can be a null mutation, which can arise frommany types of mutations including inactivating point mutations, entiregene deletions, and deletions of chromosomal segments or entirechromosomes. Specific antisense nucleic acid compounds and enzymeinhibitors, such as antibiotics, can also produce null mutant phenotype,therefore being equivalent to gene disruption.

As used herein, the term “growth-coupled” when used in reference to theproduction of a biochemical product is intended to mean that thebiosynthesis of the referenced biochemical product is produced duringthe growth phase of a microorganism. In a particular embodiment, thegrowth-coupled production can be obligatory, meaning that thebiosynthesis of the referenced biochemical is an obligatory productproduced during the growth phase of a microorganism. The term“growth-coupled” when used in reference to the consumption of abiochemical is intended to mean that the referenced biochemical isconsumed during the growth phase of a microorganism.

As used herein, the term “attenuate,” or grammatical equivalentsthereof, is intended to mean to weaken, reduce or diminish the activityor amount of an enzyme or protein. Attenuation of the activity or amountof an enzyme or protein can mimic complete disruption if the attenuationcauses the activity or amount to fall below a critical level requiredfor a given pathway to function. However, the attenuation of theactivity or amount of an enzyme or protein that mimics completedisruption for one pathway, can still be sufficient for a separatepathway to continue to function. For example, attenuation of anendogenous enzyme or protein can be sufficient to mimic the completedisruption of the same enzyme or protein for production of a fattyalcohol, fatty aldehyde or fatty acid product, but the remainingactivity or amount of enzyme or protein can still be sufficient tomaintain other pathways, such as a pathway that is critical for the hostmicrobial organism to survive, reproduce or grow. Attenuation of anenzyme or protein can also be weakening, reducing or diminishing theactivity or amount of the enzyme or protein in an amount that issufficient to increase yield of a fatty alcohol, fatty aldehyde or fattyacid, but does not necessarily mimic complete disruption of the enzymeor protein.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid canutilize either or both a heterologous or homologous encoding nucleicacid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

The NNOMOs provided herein can contain stable genetic alterations, whichrefers to microorganisms that can be cultured for greater than fivegenerations without loss of the alteration. Generally, stable geneticalterations include modifications that persist greater than 10generations, particularly stable modifications will persist more thanabout 25 generations, and more particularly, stable geneticmodifications will be greater than 50 generations, includingindefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the NNOMO. An example oforthologs exhibiting separable activities is where distinct activitieshave been separated into distinct gene products between two or morespecies or within a single species. A specific example is the separationof elastase proteolysis and plasminogen proteolysis, two types of serineprotease activity, into distinct molecules as plasminogen activator andelastase. A second example is the separation of mycoplasma 5′-3′exonuclease and Drosophila DNA polymerase III activity. The DNApolymerase from the first species can be considered an ortholog toeither or both of the exonuclease or the polymerase from the secondspecies and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the NNOMOs provided hereinhaving BDO or 4-HB biosynthetic capability, those skilled in the artwill understand with applying the teaching and guidance provided hereinto a particular species that the identification of metabolicmodifications can include identification and inclusion or inactivationof orthologs. To the extent that paralogs and/or nonorthologous genedisplacements are present in the referenced microorganism that encode anenzyme catalyzing a similar or substantially similar metabolic reaction,those skilled in the art also can utilize these evolutionally relatedgenes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

3.2 Microbial Organisms that Utilize Reducing Equivalents Produced bythe Metabolism of Methanol

Provided herein are MMPs engineered to improve the availability ofreducing equivalents, which can be used for the production of productmolecules. Exemplary product molecules include, without limitation, BDOand/or 4HB, although given the teachings and guidance provided herein,it will be recognized by one skilled in the art that any productmolecule that utilizes reducing equivalents in its production canexhibit enhanced production through the biosynthetic pathways providedherein.

Methanol is a relatively inexpensive organic feedstock that can bederived from synthesis gas components, CO and H₂, via catalysis.Methanol can be used as a source of reducing equivalents to increase themolar yield of product molecules from carbohydrates.

BDO is a valuable chemical for the production of high performancepolymers, solvents, and fine chemicals. It is the basis for producingother high value chemicals such as tetrahydrofuran (THF) andgamma-butyrolactone (GBL). The value chain is comprised of three mainsegments including: (1) polymers, (2) THF derivatives, and (3) GBLderivatives. In the case of polymers, BDO is a comonomer forpolybutylene terephthalate (PBT) production. PBT is a medium performanceengineering thermoplastic used in automotive, electrical, water systems,and small appliance applications. Conversion to THF, and subsequently topolytetramethylene ether glycol (PTMEG), provides an intermediate usedto manufacture spandex products such as LYCRA® fibers. PTMEG is alsocombined with BDO in the production of specialty polyester ethers(COPE). COPEs are high modulus elastomers with excellent mechanicalproperties and oil/environmental resistance, allowing them to operate athigh and low temperature extremes. PTMEG and BDO also make thermoplasticpolyurethanes processed on standard thermoplastic extrusion,calendaring, and molding equipment, and are characterized by theiroutstanding toughness and abrasion resistance. The GBL produced from BDOprovides the feedstock for making pyrrolidones, as well as serving theagrochemical market. The pyrrolidones are used as high performancesolvents for extraction processes of increasing use, including forexample, in the electronics industry and in pharmaceutical production.Accordingly, provided herein is bioderived BDO produced according to themethods described herein and biobased products comprising or obtainedusing the bioderived BDO. The biobased product can comprise a polymer,THF or a THF derivative, or GBL or a GBL derivative; or the biobasedproduct can comprise a polymer, a plastic, elastic fiber, polyurethane,polyester, polyhydroxyalkanoate, poly-4-HB, co-polymer of poly-4-HB,poly(tetramethylene ether) glycol, polyurethane-polyurea copolymer,spandex, elastane, Lycra™, or nylon; or the biobased product cancomprise polybutylene terephthalate (PBT) polymer; or the biobasedproduct can comprise a PBT polymer that comprises a resin, a fiber, abead, a granule, a pellet, a chip, a plastic, a polyester, athermoplastic polyester, a molded article, an injection-molded article,an injection-molded part, an automotive part, an extrusion resin, anelectrical part and a casing, optionally where the biobased product isreinforced or filled, for example glass-filled or mineral-filled; or thebiobased product is THF or a THF derivative, and the THF derivative ispolytetramethylene ether glycol (PTMEG), a polyester ether (COPE) or athermoplastic polyurethane or a fiber; or the biobased product comprisesGBL or a GBL derivative and the GBL derivative is a pyrrolidone. Thebiobased product can comprise at least 5%, at least 10%, at least 20%,at least 30%, at least 40% or at least 50% bioderived BDO. The biobasedproduct can comprises a portion of said bioderived BDO as a repeatingunit. The biobased product can be a molded product obtained by moldingthe biobased product.

BDO is produced by two main petrochemical routes with a few additionalroutes also in commercial operation. One route involves reactingacetylene with formaldehyde, followed by hydrogenation. More recently,BDO processes involving butane or butadiene oxidation to maleicanhydride, followed by hydrogenation have been introduced. BDO is usedalmost exclusively as an intermediate to synthesize other chemicals andpolymers. Thus, there exists a need for the development of methods foreffectively producing commercial quantities of BDO.

In numerous engineered pathways, realization of maximum product yieldsbased on carbohydrate feedstock is hampered by insufficient reducingequivalents or by loss of reducing equivalents to byproducts. Methanolis a relatively inexpensive organic feedstock that can be used togenerate reducing equivalents by employing one or more methanolmetabolic enzymes as shown in FIG. 1. The reducing equivalents producedby the metabolism of methanol by one or more of the MMPs can then beused to power the glucose to BDO production pathways, for example, asshown in FIG. 2.

The product yields per C-mol of substrate of microbial cellssynthesizing reduced fermentation products such as BDO and 4-HB arelimited by insufficient reducing equivalents in the carbohydratefeedstock. Reducing equivalents, or electrons, can be extracted frommethanol using one or more of the enzymes described in FIG. 1. Thereducing equivalents are then passed to acceptors such as oxidizedferredoxins, oxidized quinones, oxidized cytochromes, NAD(P)+, water, orhydrogen peroxide to form reduced ferredoxin, reduced quinones, reducedcytochromes, NAD(P)H, H₂, or water, respectively. Reduced ferredoxin,reduced quinones and NAD(P)H are particularly useful as they can serveas redox carriers for various Wood-Ljungdahl pathway, reductive TCAcycle, or product pathway enzymes.

Specific examples of how additional redox availability from methanol canimprove the yield of reduced products such as succinate, 4-HB, and BDOare shown.

The maximum theoretical yield of BDO via the pathway shown in FIG. 2supplemented with the reactions of the oxidative TCA cycle (e.g.,citrate synthase, aconitase, isocitrate dehydrogenase,alpha-ketoglutarate dehydrogenase) is 1.09 mol/mol.

1 C₆H₁₂O₆→1.09 C₄H₁₀O₂+1.64 CO₂+0.55 H₂O

When both feedstocks of sugar and methanol are available, the methanolcan be utilized to generate reducing equivalents by employing one ormore of the enzymes shown in FIG. 1. The reducing equivalents generatedfrom methanol can be utilized to power the glucose to BDO productionpathways, e.g., as shown in FIG. 2. Theoretically, all carbons inglucose will be conserved, thus resulting in a maximal theoretical yieldto produce BDO from glucose at 2 mol BDO per mol of glucose under eitheraerobic or anaerobic conditions as shown in FIG. 2:

10 CH₃OH+3 C₆H₁₂O₆=6 C₄H₁₀O₂+8 H₂O+4 CO₂

In a similar manner, the maximum theoretical yields of succinate and4-HB can reach 2 mol/mol glucose using the reactions shown in FIGS. 1and 2.

C₆H₁₂O₆+0.667 CH₃OH+1.333 CO₂→2 C₄H₆O₄+1.333 H₂O

C₆H₁₂O₆+2 CH₃OH→2 C₄H₈O₃+2 H₂O

In a first aspect, provided herein is a NNOMO having a MMP, wherein saidorganism comprises at least one exogenous nucleic acid encoding a MMPE.In certain embodiments, the MMPE is expressed in a sufficient amount toenhance the availability of reducing equivalents in the presence ofmethanol. In other embodiments, the MMPE is expressed in a sufficientamount to convert methanol to formaldehyde. In certain embodiments, theMMP comprises one or more enzymes selected from the group consisting ofan EM1; an EM2; an EM3; an EM4; an EM5; an EM6; an EM15; an EM16; anEM8; an EM9; an EM10; an EM11; an EM12; an EM13; and an EM14. Suchorganisms advantageously allow for the production of reducingequivalents, which can then be used by the organism for the productionof BDO or 4-HB using any one of the pathways provided herein.

In certain embodiments, the MMP comprises 1A, 1B, 1C, 1D, 1E, 1F, 1G,1H, 1I, 1J, 1K, 1L, 1M, 1N, or 10 or any combination of 1A, 1B, 1C, 1D,1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, and 1O, thereof, wherein 1A isan EM1; 1B is an EM2; 1C is an EM3; 1D is an EM4; 1E is an EM5; 1F is anEM6; 1G is an EM15; 1H is an EM16, 1I is an EM8; 1J is an EM9; 1K is anEM10; 1L is an EM11; 1M is an EM12; 1N is an EM13; and 1O is an EM14. Insome embodiments, 1K is spontaneous. In other embodiments, 1K is anEM10. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis an EM12.

In one embodiment, the MMP comprises 1A. In another embodiment, the MMPcomprises 1B. In another embodiment, the MMP comprises 1C. In yetanother embodiment, the MMP comprises 1D. In one embodiment, the MMPcomprises 1E. In another embodiment, the MMP comprises 1F. In anotherembodiment, the MMP comprises 1G. In yet another embodiment, the MMPcomprises 1H. In one embodiment, the MMP comprises 1I. In anotherembodiment, the MMP comprises 1J. In another embodiment, the MMPcomprises 1K. In yet another embodiment, the MMP comprises 1L. In yetanother embodiment, the MMP comprises 1M. In another embodiment, the MMPcomprises 1N. In yet another embodiment, the MMP comprises 1O. Anycombination of two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen or fifteen MMPEs 1A, 1B, 1C, 1D, 1E,1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, and 1O is also contemplated.

In some embodiments, the MMP is a MMP depicted in FIG. 1.

In one aspect, provided herein is a NNOMO having a MMP, wherein saidorganism comprises at least one exogenous nucleic acid encoding a MMPEexpressed in a sufficient amount to enhance the availability of reducingequivalents in the presence of methanol, wherein said MMP comprises: (i)1A and 1B, (ii) 1J; or (iii) 1J and 1K. In one embodiment, the MMPcomprises 1A and 1B. In another embodiment, the MMP comprises 1J. In oneembodiment, the MMP comprises 1J and 1K. In certain embodiments, the MMPcomprises 1A, 1B, 1C, 1D, and 1E. In some embodiments, the MMP comprises1A, 1B, 1C, 1D and 1F. In some embodiments, the MMP comprises 1J, 1C, 1Dand 1E. In one embodiment, the MMP comprises 1J, 1C, 1D and 1F. Inanother embodiment, the MMP comprises 1J and 1L. In yet anotherembodiment, the MMP comprises 1J, 1M, 1N and 1O. In certain embodiments,the MMP comprises 1J, 1N and 1O. In some embodiments, the MMP comprises1J, 1K, 1C, 1D and 1E. In one embodiment, the MMP comprises 1J, 1K, 1C,1D and 1F. In some embodiments, 1K is spontaneous. In other embodiments,1K is an EM10. In some embodiments, 1M is spontaneous. In otherembodiments, 1M is an EM12.

In certain embodiments, the MMP comprises 1I. In certain embodiments,the MMP comprises 1A, 1B, 1C, 1D, 1E and 1I. In some embodiments, theMMP comprises 1A, 1B, 1C, 1D, 1F and 1I. In some embodiments, the MMPcomprises 1J, 1C, 1D, 1E and 1I. In one embodiment, the MMP comprises1J, 1C, 1D, 1F and 1I. In another embodiment, the MMP comprises 1J, 1Land 1I. In yet another embodiment, the MMP comprises 1J, 1M, 1N, 1O and1I. In certain embodiments, the MMP comprises 1J, 1N, 1O and 1I. In someembodiments, the MMP comprises 1J, 1K, 1C, 1D, 1E and 1I. In oneembodiment, the MMP comprises 1J, 1K, 1C, 1D, 1F and 1I. In someembodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. Insome embodiments, 1M is spontaneous. In other embodiments, 1M is anEM12.

In certain embodiments, the MMP comprises 1G. In certain embodiments,the MMP comprises 1A, 1B, 1C, 1D, 1E and 1G. In some embodiments, theMMP comprises 1A, 1B, 1C, 1D, 1F and 1G. In some embodiments, the MMPcomprises 1J, 1C, 1D, 1E and 1G. In one embodiment, the MMP comprises1J, 1C, 1D, 1F and 1G. In another embodiment, the MMP comprises 1J, 1Land 1G. In yet another embodiment, the MMP comprises 1J, 1M, 1N, 1O and1G. In certain embodiments, the MMP comprises 1J, 1N, 1O and 1G. In someembodiments, the MMP comprises 1J, 1K, 1C, 1D, 1E and 1G. In oneembodiment, the MMP comprises 1J, 1K, 1C, 1D, 1F and 1G. In someembodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. Insome embodiments, 1M is spontaneous. In other embodiments, 1M is anEM12.

In certain embodiments, the MMP comprises 1G and 1H. In certainembodiments, the MMP comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H. In someembodiments, the MMP comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H. In someembodiments, the MMP comprises 1J, 1C, 1D, 1E, 1G and 1H. In oneembodiment, the MMP comprises 1J, 1C, 1D, 1F, 1G and 1H. In anotherembodiment, the MMP comprises 1J, 1L, 1G and 1H. In yet anotherembodiment, the MMP comprises 1J, 1M, 1N, 1O, 1G and 1H. In certainembodiments, the MMP comprises 1J, 1N, 1O, 1G and 1H. In someembodiments, the MMP comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H. In oneembodiment, the MMP comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H. In someembodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. Insome embodiments, 1M is spontaneous. In other embodiments, 1M is anEM12.

In certain embodiments, the formation of 5-hydroxymethylglutathione fromformaldehyde is spontaneous (see, e.g., FIG. 1, step M). In someembodiments, the formation of 5-hydroxymethylglutathione fromformaldehyde is catalyzed by an EM12 (see, e.g., FIG. 1, step M). Incertain embodiments, the formation of methylene-THF from formaldehyde isspontaneous (see, e.g., FIG. 1, step K). In certain embodiments, theformation of methylene-THF from formaldehyde is catalyzed by an EM10(see, e.g., FIG. 1, step K).

In certain embodiments, the organism comprises two, three, four, five,six or seven exogenous nucleic acids, each encoding a MMPE. In certainembodiments, the organism comprises two exogenous nucleic acids, eachencoding a MMPE. In certain embodiments, the organism comprises threeexogenous nucleic acids, each encoding a MMPE. In certain embodiments,the organism comprises four exogenous nucleic acids, each encoding aMMPE. In certain embodiments, the organism comprises five exogenousnucleic acids, each encoding a MMPE. In certain embodiments, theorganism comprises six exogenous nucleic acids, each encoding a MMPE. Incertain embodiments, the organism comprises seven exogenous nucleicacids, each encoding a MMPE.

Any non-naturally occurring eukaryotic organism comprising a MMP andengineered to comprise a MMPE, such as those provided herein, can beengineered to further comprise one or more BDOP enzymes (BDOPEs).

In certain embodiments, the NNOMO further comprises a BDOP, wherein saidorganism comprises at least one exogenous nucleic acid encoding a BDOPEexpressed in a sufficient amount to produce BDO. In certain embodiments,the BDOPE is selected from the group consisting of an EB1 or an EB2A; anEB3; an EB4; a EB5; an EB6, an EB7; an EB8; an EB9; an EB10; an EB11 oran EB12; an EB13; an EB14, and an EB15.

In some embodiments, the NNOMOs having a BDOP include a set of BDOPEs.

Enzymes, genes and methods for engineering pathways from succinate andsuccinyl-CoA to various products, such as BDO, into a microorganism, arenow known in the art (see, e.g., U.S. Publ. No. 2011/0201089). A set ofBDOPEs represents a group of enzymes that can convert succinate to BDOas shown in FIG. 2. The additional reducing equivalents obtained fromthe MMPs, as disclosed herein, improve the yields of all these productswhen utilizing carbohydrate-based feedstock. For example, BDO can beproduced from succinyl-CoA via previously disclosed pathways (see forexample, Burk et al., WO 2008/115840). Exemplary enzymes for theconversion succinyl-CoA to BDO include EB3 (FIG. 2, Step B), EB4 (FIG.2, Step C), EB5 (FIG. 2, Step D), EB6 (FIG. 2, Step E), EB7 (FIG. 2,Step F), EB8 (FIG. 2, Step G), EB10 (FIG. 1, Step I), EB11 (FIG. 2, StepJ), EB12 (FIG. 2, Step J), EB14 (FIG. 2, Step L), EB13 (FIG. 2, Step K),and EB15 (FIG. 2, Step M). EB9 (FIG. 2, Step H) can be additionallyuseful in converting succinate directly to the BDOP intermediate,succinate semialdehyde.

In another aspect, provided herein is a NNOMO, comprising (1) a MMP,wherein said organism comprises at least one exogenous nucleic acidencoding a MMPE in a sufficient amount to enhance the availability ofreducing equivalents in the presence of methanol; and (2) a BDOP,comprising at least one exogenous nucleic acid encoding a BDOPEexpressed in a sufficient amount to produce BDO. In one embodiment, theat least one exogenous nucleic acid encoding the MMPE enhances theavailability of reducing equivalents in the presence of methanol in asufficient amount to increase the amount of BDO produced by thenon-naturally microbial organism. In some embodiments, the MMP comprisesany of the various combinations of MMPEs described above or elsewhereherein.

In certain embodiments, (1) the MMP comprises: 1A, 1B, 1C, 1D, 1E, 1F,1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or any combination of 1A, 1B, 1C,1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O, thereof, wherein 1Ais an EM1; 1B is an EM2; 1C is an EM3; 1D is an EM4; 1E is an EM5; 1F isan EM6; 1G is an EM15; 1H is an EM16, 1I is an EM8; 1J is an EM9; 1K isspontaneous or EM10; 1L is an EM11; 1M is spontaneous or an EM12; 1N isan EM13 and 1O is EM14; and (2) the BDOP comprises 2A, 2B, 2C, 2D, 2E,2F, 2G, 2H, 2I, 2J, 2K, 2L or 2M or any combination of 2A, 2B, 2C, 2D,2E, 2F, 2G, 2H, 2I, 2J, 2K, 2L or 2M, wherein 2A is an EB1 or an EB2A;2B is an EB3; 2C is an EB4; 2D is an EB5; 2E is an EB6; 2F is an EB7; 2Gis an EB8; 2H a is EB9; 2I a is EB10; 2J is an EB11 or EB12; 2K is anEB13; 2L is an EB14; and 2M is an EB15. In some embodiments, 2A is anEB1. In some embodiments, 2A is an EB2A. In some embodiments, 2J is anEB11. In some embodiments, 2J is an EB12. In some embodiments, 1K isspontaneous. In other embodiments, 1K is an EM10. In some embodiments,1M is spontaneous. In other embodiments, 1M is an EM12. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12

In one embodiment, the BDOP comprises 2A. In another embodiment, theBDOP comprises 2B. In an embodiment, the BDOP comprises 2C. In anotherembodiment, the BDOP comprises 2D. In one embodiment, the BDOP comprises2E. In yet another embodiment, the BDOP comprises 2F. In someembodiments, the BDOP comprises 2G. In other embodiments, the BDOPcomprises 2H. In another embodiment, the BDOP comprises 2I. In oneembodiment, the BDOP comprises 2J. In one embodiment, the BDOP comprises2K. In another embodiment, the BDOP comprises 2L. In an embodiment, theBDOP comprises 2M. Any combination of two, three, four, five, six,seven, eight, nine, ten, eleven, twelve or thirteen BDOPEs 2A, 2B, 2C,2D, 2E, 2F, 2G, 2H, 2I, 2J, 2K, 2L and 2M is also contemplated. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12

In some embodiments, the MMP is a MMP depicted in FIG. 1, and the BDOPis a BDOP depicted in FIG. 2. In certain embodiments, the formation of5-hydroxymethylglutathione from formaldehyde is spontaneous (see, e.g.,FIG. 1, step M). In some embodiments, the formation of5-hydroxymethylglutathione from formaldehyde is catalyzed by an EM12(see, e.g., FIG. 1, step M). In certain embodiments, the formation ofmethylene-THF from formaldehyde is spontaneous (see, e.g., FIG. 1, stepK). In certain embodiments, the formation of methylene-THF fromformaldehyde is catalyzed by an EM10 (see, e.g., FIG. 1, step K).

Exemplary sets of BDOPEs to convert succinate to BDO, according to FIG.2, include 2A, 2B, 2C, 2D, 2E, 2F, and 2G; 2A, 2B, 2C, 2J, 2F, and 2G;2A, 2B, 2C, 2J, and 2M; 2A, 2B, 2C, 2D, 2E, and 2M; 2A, 2B, 2C, 2K, and2G; 2A, 2B, 2C, 2D, 2L, and 2G; 2A, 2I, 2D, 2E, 2F, and 2G; 2A, 2I, 2D,2E, and 2M; 2A, 2I, 2J, 2F, and 2G; 2A, 2I, 2J, and 2M; 2A, 2I, 2K, and2G; 2A, 2I, 2D, 2L and 2G; 2H, 2C, 2D, 2E, 2F, and 2G; 2H, 2C, 2J, 2F,and 2G; 2H, 2C, 2J, and 2M; 2H, 2C, 2D, 2E, and 2M; 2H, 2C, 2K, and 2G;and 2H, 2C, 2D, 2L, and 2G. In one embodiment, 2J is an EB11. In anotherembodiment, 2J is an EB12.

In one embodiment, the BDOP comprises 2B, 2C, 2D, 2E, 2F, and 2G. Inanother embodiment, the BDOP comprises 2B, 2C, 2J, 2F, and 2G. Inanother embodiment, the BDOP comprises 2B, 2C, 2J, and 2M. In yetembodiment, the BDOP comprises 2B, 2C, 2D, 2E, and 2M. In oneembodiment, the BDOP comprises 2B, 2C, 2K, and 2G. In anotherembodiment, the BDOP comprises 2B, 2C, 2D, 2L, and 2G. In anotherembodiment, the BDOP comprises 2I, 2D, 2E, 2F, and 2G. In yet anotherembodiment, the BDOP 2I, 2D, 2E, and 2M. In one embodiment, the BDOPcomprises 2I, 2J, 2F, and 2G. In another embodiment, the BDOP comprises2I, 2J, and 2M. In yet another embodiment, the BDOP comprises 2I, 2K,and 2G. In one embodiment, the BDOP comprises 2I, 2D, 2L and 2G. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12

In certain embodiments, the BDOP further comprises 2A. In oneembodiment, the BDOP comprises 2A, 2B, 2C, 2D, 2E, 2F, and 2G. Inanother embodiment, the BDOP comprises 2A, 2B, 2C, 2J, 2F, and 2G. Inanother embodiment, the BDOP comprises 2A, 2B, 2C, 2J, and 2M. In yetembodiment, the BDOP comprises 2A, 2B, 2C, 2D, 2E, and 2M. In oneembodiment, the BDOP comprises 2A, 2B, 2C, 2K, and 2G. In anotherembodiment, the BDOP comprises 2A, 2B, 2C, 2D, 2L, and 2G. In anotherembodiment, the BDOP comprises 2A, 2I, 2D, 2E, 2F, and 2G. In yetanother embodiment, the BDOP 2A, 2I, 2D, 2E, and 2M. In one embodiment,the BDOP comprises 2A, 2I, 2J, 2F, and 2G. In another embodiment, theBDOP comprises 2A, 2I, 2J, and 2M. In yet another embodiment, the BDOPcomprises 2A, 2I, 2K, and 2G. In one embodiment, the BDOP comprises 2A,2I, 2D, 2L and 2G. In one embodiment, 2J is an EB11. In anotherembodiment, 2J is an EB12

In another embodiment, the BDOP comprises 2H, 2C, 2D, 2E, 2F, and 2G. Inanother embodiment, the BDOP comprises 2H, 2C, 2J, 2F, and 2G. In yetanother embodiment, the BDOP comprises 2H, 2C, 2J, and 2M. In oneembodiment, the BDOP comprises 2H, 2C, 2D, 2E, and 2M. In anotherembodiment, the BDOP comprises 2H, 2C, 2K, and 2G. In yet anotherembodiment, the BDOP comprises and 2H, 2C, 2D, 2L, and 2G. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12

In one embodiment, (1) the MMP comprises: (i) 1A and 1B, (ii) 1J; or(iii) 1J and 1K; and (2) the BDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and2G; (b) 2B, 2C, 2J, 2F, and 2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D,2E, and 2M; (e) 2B, 2C, 2K, and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2I,2D, 2E, 2F, and 2G; (h) 2I, 2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j)2I, 2J, and 2M; (k) 2I, 2K, and 2G; or (l) 2I, 2D, 2L and 2G. In someembodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. Insome embodiments, 1M is spontaneous. In other embodiments, 1M is anEM12. In one embodiment, 2J is an EB11. In another embodiment, 2J is anEB12.

In another embodiment, (1) the MMP comprises: (i) 1A and 1B, (ii) 1J; or(iii) 1J and 1K; and (2) the BDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F,and 2G; (b) 2A, 2B, 2C, 2J, 2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d)2A, 2B, 2C, 2D, 2E, and 2M; (e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C,2D, 2L, and 2G; (g) 2A, 2I, 2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and2M; (i) 2A, 2I, 2J, 2F, and 2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K,and 2G; (l) 2A, 2I, 2D, 2L and 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n)2H, 2C, 2J, 2F, and 2G; (o) 2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and2M; (q) 2H, 2C, 2K, and 2G; or (r) 2H, 2C, 2D, 2L, and 2G. In someembodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. Insome embodiments, 1M is spontaneous. In other embodiments, 1M is anEM12. In one embodiment, 2J is an EB11. In another embodiment, 2J is anEB12.

In one embodiment, (1) the MMP comprises 1A and 1B; and (2) the BDOPcomprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F, and 2G;(c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C, 2K, and2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2I, 2D, 2E, 2F, and 2G; (h) 2I, 2D,2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I, 2K, and2G; or (l) 2I, 2D, 2L and 2G. In some embodiments, 1K is spontaneous. Inother embodiments, 1K is an EM10. In some embodiments, 1M isspontaneous. In other embodiments, 1M is an EM12. In one embodiment, 2Jis an EB11. In another embodiment, 2J is an EB12.

In another embodiment, (1) the MMP comprises 1A and 1B; and (2) the BDOPcomprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C, 2J, 2F,and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and 2M; (e)2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A, 2I, 2D,2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F, and 2G;(j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D, 2L and2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G; (o) 2H,2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and 2G; or(r) 2H, 2C, 2D, 2L, and 2G. In one embodiment, 2J is an EB11. In anotherembodiment, 2J is an EB12.

In one embodiment, (1) the MMP comprises 1J; and (2) the BDOP comprises(a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F, and 2G; (c) 2B, 2C,2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C, 2K, and 2G; (f) 2B,2C, 2D, 2L, and 2G; (g) 2I, 2D, 2E, 2F, and 2G; (h) 2i, 2D, 2E, and 2M;(i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I, 2K, and 2G; or (l)2I, 2D, 2L and 2G. In one embodiment, 2J is an EB11. In anotherembodiment, 2J is an EB12.

In another embodiment, (1) the MMP comprises 1J; and (2) the BDOPcomprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C, 2J, 2F,and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and 2M; (e)2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A, 2I, 2D,2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F, and 2G;(j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D, 2L and2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G; (o) 2H,2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and 2G; or(r) 2H, 2C, 2D, 2L, and 2G. In one embodiment, 2J is an EB11. In anotherembodiment, 2J is an EB12.

In one embodiment, (1) the MMP comprises 1J and 1K; and (2) the BDOPcomprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F, and 2G;(c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C, 2K, and2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2I, 2D, 2E, 2F, and 2G; (h) 2I, 2D,2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I, 2K, and2G; or (l) 2I, 2D, 2L and 2G. In some embodiments, 1K is spontaneous. Inother embodiments, 1K is an EM10. In one embodiment, 2J is an EB11. Inanother embodiment, 2J is an EB12.

In one embodiment, (1) the MMP comprises 1J and 1K; and (2) the BDOPcomprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C, 2J, 2F,and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and 2M; (e)2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A, 2I, 2D,2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F, and 2G;(j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D, 2L and2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G; (o) 2H,2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and 2G; or(r) 2H, 2C, 2D, 2L, and 2G. In some embodiments, 1K is spontaneous. Inother embodiments, 1K is an EM10. In one embodiment, 2J is an EB11. Inanother embodiment, 2J is an EB12.

In certain embodiments, (1) the MMP comprises 1A, 1B, 1C, 1D, and 1E;and (2) the BDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C,2J, 2F, and 2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e)2B, 2C, 2K, and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2i, 2D, 2E, 2F, and2G; (h) 2I, 2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M;(k) 2I, 2K, and 2G; or (l) 2I, 2D, 2L and 2G. In certain embodiments,the MMP further comprises 1I. In some embodiments, the MMP furthercomprises 1G. In other embodiments, the MMP further comprises 1G and 1H.In one embodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In certain embodiments, (1) the MMP comprises 1A, 1B, 1C, 1D, and 1E;and (2) the BDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A,2B, 2C, 2J, 2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D,2E, and 2M; (e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G;(g) 2A, 2I, 2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I,2J, 2F, and 2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A,2I, 2D, 2L and 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F,and 2G; (o) 2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C,2K, and 2G; or (r) 2H, 2C, 2D, 2L, and 2G. In certain embodiments, theMMP further comprises 1I. In some embodiments, the MMP further comprises1G. In other embodiments, the MMP further comprises 1G and 1H. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In some embodiments. (1) the MMP comprises 1A, 1B, 1C, 1D and 1F; and(2) the BDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J,2F, and 2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B,2C, 2K, and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2i, 2D, 2E, 2F, and 2G;(h) 2I, 2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k)2I, 2K, and 2G; or (l) 2I, 2D, 2L and 2G. In certain embodiments, theMMP further comprises 1I. In some embodiments, the MMP further comprisesIG. In other embodiments, the MMP further comprises 1G and 1H. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In some embodiments. (1) the MMP comprises 1A, 1B, 1C, 1D and 1F; and(2) the BDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B,2C, 2J, 2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E,and 2M; (e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g)2A, 2I, 2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J,2F, and 2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I,2D, 2L and 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and2G; (o) 2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K,and 2G; or (r) 2H, 2C, 2D, 2L, and 2G. In certain embodiments, the MMPfurther comprises 1I. In some embodiments, the MMP further comprises IG.In other embodiments, the MMP further comprises 1G and 1H. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In some embodiments, (1) the MMP comprises 1J, 1C, 1D and 1E; and (2)the BDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F,and 2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C,2K, and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2I, 2D, 2E, 2F, and 2G; (h)2I, 2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I,2K, and 2G; or (l) 2I, 2D, 2L and 2G. In certain embodiments, the MMPfurther comprises 1I. In some embodiments, the MMP further comprises IG.In other embodiments, the MMP further comprises 1G and 1H. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In some embodiments, (1) the MMP comprises 1J, 1C, 1D and 1E; and (2)the BDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C,2J, 2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and2M; (e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A,2I, 2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F,and 2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D,2L and 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G;(o) 2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and2G; or (r) 2H, 2C, 2D, 2L, and 2G. In certain embodiments, the MMPfurther comprises 1I. In some embodiments, the MMP further comprises IG.In other embodiments, the MMP further comprises 1G and 1H. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In one embodiment, (1) the MMP comprises 1A, 1B, and 1C; and (2) theBDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F, and2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C, 2K,and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2I, 2D, 2E, 2F, and 2G; (h) 2I,2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I, 2K,and 2G; or (l) 2I, 2D, 2L and 2G. In one embodiment, 2J is an EB11. Inanother embodiment, 2J is an EB12.

In one embodiment, (1) the MMP comprises 1A, 1B, and 1C; and (2) theBDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C, 2J,2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and 2M;(e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A, 2I,2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F, and2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D, 2Land 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G; (o)2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and 2G;or (r) 2H, 2C, 2D, 2L, and 2G. In one embodiment, 2J is an EB11. Inanother embodiment, 2J is an EB12.

In yet another embodiment, (1) the MMP comprises 1J, 1M, and 1N; and (2)the BDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F,and 2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C,2K, and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2i, 2D, 2E, 2F, and 2G; (h)2I, 2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I,2K, and 2G; or (l) 2I, 2D, 2L and 2G. In some embodiments, 1M isspontaneous. In other embodiments, 1M is an EM12. In one embodiment, 2Jis an EB11. In another embodiment, 2J is an EB12.

In yet another embodiment, (1) the MMP comprises 1J, 1M, and 1N; and (2)the BDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C,2J, 2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and2M; (e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A,2I, 2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F,and 2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D,2L and 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G;(o) 2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and2G; or (r) 2H, 2C, 2D, 2L, and 2G. In some embodiments, 1M isspontaneous. In other embodiments, 1M is an EM12. In one embodiment, 2Jis an EB11. In another embodiment, 2J is an EB12.

In one embodiment, (1) the MMP comprises 1J, 1C, 1D and 1F; and (2) theBDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F, and2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C, 2K,and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2I, 2D, 2E, 2F, and 2G; (h) 2I,2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I, 2K,and 2G; or (l) 2I, 2D, 2L and 2G. In certain embodiments, the MMPfurther comprises 1I. In some embodiments, the MMP further comprises IG.In other embodiments, the MMP further comprises 1G and 1H. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In one embodiment, (1) the MMP comprises 1J, 1C, 1D and 1F; and (2) theBDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C, 2J,2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and 2M;(e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A, 2I,2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F, and2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D, 2Land 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G; (o)2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and 2G;or (r) 2H, 2C, 2D, 2L, and 2G. In certain embodiments, the MMP furthercomprises 1I. In some embodiments, the MMP further comprises IG. Inother embodiments, the MMP further comprises 1G and 1H. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In another embodiment, (1) the MMP comprises 1J and 1L; and (2) the BDOPcomprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F, and 2G;(c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C, 2K, and2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2I, 2D, 2E, 2F, and 2G; (h) 2I, 2D,2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I, 2K, and2G; or (l) 2I, 2D, 2L and 2G. In certain embodiments, the MMP furthercomprises 1I. In some embodiments, the MMP further comprises IG. Inother embodiments, the MMP further comprises 1G and 1H. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In another embodiment, (1) the MMP comprises 1J and 1L; and (2) the BDOPcomprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C, 2J, 2F,and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and 2M; (e)2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A, 2I, 2D,2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F, and 2G;(j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D, 2L and2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G; (o) 2H,2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and 2G; or(r) 2H, 2C, 2D, 2L, and 2G. In certain embodiments, the MMP furthercomprises 1I. In some embodiments, the MMP further comprises IG. Inother embodiments, the MMP further comprises 1G and 1H. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In yet another embodiment, (1) the MMP comprises 1J, 1M, 1N and 1O; and(2) the BDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J,2F, and 2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B,2C, 2K, and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2i, 2D, 2E, 2F, and 2G;(h) 2I, 2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k)2I, 2K, and 2G; or (l) 2I, 2D, 2L and 2G. In certain embodiments, theMMP further comprises 1I. In some embodiments, the MMP further comprisesIG. In other embodiments, the MMP further comprises 1G and 1H. In someembodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. Inone embodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In yet another embodiment, (1) the MMP comprises 1J, 1M, 1N and 1O; and(2) the BDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B,2C, 2J, 2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E,and 2M; (e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g)2A, 2I, 2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J,2F, and 2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I,2D, 2L and 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and2G; (o) 2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K,and 2G; or (r) 2H, 2C, 2D, 2L, and 2G. In certain embodiments, the MMPfurther comprises 1I. In some embodiments, the MMP further comprises IG.In other embodiments, the MMP further comprises 1G and 1H. In someembodiments, 1M is spontaneous. In other embodiments, 1M is an EM12. Inone embodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In certain embodiments, (1) the MMP comprises 1J, 1N and 1O; and (2) theBDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F, and2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C, 2K,and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2I, 2D, 2E, 2F, and 2G; (h) 2I,2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I, 2K,and 2G; or (l) 2I, 2D, 2L and 2G. In certain embodiments, the MMPfurther comprises 1I. In some embodiments, the MMP further comprises IG.In other embodiments, the MMP further comprises 1G and 1H. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In certain embodiments, (1) the MMP comprises 1J, 1N and 1O; and (2) theBDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C, 2J,2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and 2M;(e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A, 2I,2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F, and2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D, 2Land 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G; (o)2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and 2G;or (r) 2H, 2C, 2D, 2L, and 2G. In certain embodiments, the MMP furthercomprises 1I. In some embodiments, the MMP further comprises IG. Inother embodiments, the MMP further comprises 1G and 1H. In oneembodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In some embodiments, (1) the MMP comprises 1J, 1K, 1C, 1D and 1E; and(2) the BDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J,2F, and 2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B,2C, 2K, and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2i, 2D, 2E, 2F, and 2G;(h) 2I, 2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k)2I, 2K, and 2G; or (l) 2I, 2D, 2L and 2G. In certain embodiments, theMMP further comprises 1I. In some embodiments, the MMP further comprisesIG. In other embodiments, the MMP further comprises 1G and 1H. In someembodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. Inone embodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In some embodiments, (1) the MMP comprises 1J, 1K, 1C, 1D and 1E; and(2) the BDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B,2C, 2J, 2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E,and 2M; (e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g)2A, 2I, 2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J,2F, and 2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I,2D, 2L and 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and2G; (o) 2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K,and 2G; or (r) 2H, 2C, 2D, 2L, and 2G. In certain embodiments, the MMPfurther comprises 1I. In some embodiments, the MMP further comprises IG.In other embodiments, the MMP further comprises 1G and 1H. In someembodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. Inone embodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In one embodiment, (1) the MMP comprises 1J, 1K, 1C, 1D and 1F; and (2)the BDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F,and 2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C,2K, and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2i, 2D, 2E, 2F, and 2G; (h)2I, 2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I,2K, and 2G; or (l) 2I, 2D, 2L and 2G. In certain embodiments, the MMPfurther comprises 1I. In some embodiments, the MMP further comprises IG.In other embodiments, the MMP further comprises 1G and 1H. In someembodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. Inone embodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In one embodiment, (1) the MMP comprises 1J, 1K, 1C, 1D and 1F; and (2)the BDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C,2J, 2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and2M; (e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A,2I, 2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F,and 2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D,2L and 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G;(o) 2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and2G; or (r) 2H, 2C, 2D, 2L, and 2G. In certain embodiments, the MMPfurther comprises 1I. In some embodiments, the MMP further comprises IG.In other embodiments, the MMP further comprises 1G and 1H. In someembodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. Inone embodiment, 2J is an EB11. In another embodiment, 2J is an EB12.

In one embodiment, (1) the MMP comprises 1A, 1B, and 1C; and (2) theBDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F, and2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C, 2K,and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2I, 2D, 2E, 2F, and 2G; (h) 2I,2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I, 2K,and 2G; or (l) 2I, 2D, 2L and 2G. In one embodiment, 2J is an EB11. Inanother embodiment, 2J is an EB12.

In one embodiment, (1) the MMP comprises 1A, 1B, and 1C; and (2) theBDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C, 2J,2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and 2M;(e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A, 2I,2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F, and2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D, 2Land 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G; (o)2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and 2G;or (r) 2H, 2C, 2D, 2L, and 2G. In one embodiment, 2J is an EB11. Inanother embodiment, 2J is an EB12.

In yet another embodiment, (1) the MMP comprises 1J, 1M, and 1N; and (2)the BDOP comprises (a) 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2B, 2C, 2J, 2F,and 2G; (c) 2B, 2C, 2J, and 2M; (d) 2B, 2C, 2D, 2E, and 2M; (e) 2B, 2C,2K, and 2G; (f) 2B, 2C, 2D, 2L, and 2G; (g) 2i, 2D, 2E, 2F, and 2G; (h)2I, 2D, 2E, and 2M; (i) 2I, 2J, 2F, and 2G; (j) 2I, 2J, and 2M; (k) 2I,2K, and 2G; or (l) 2I, 2D, 2L and 2G. In some embodiments, 1M isspontaneous. In other embodiments, 1M is an EM12. In one embodiment, 2Jis an EB11. In another embodiment, 2J is an EB12.

In yet another embodiment, (1) the MMP comprises 1J, 1M, and 1N; and (2)the BDOP comprises (a) 2A, 2B, 2C, 2D, 2E, 2F, and 2G; (b) 2A, 2B, 2C,2J, 2F, and 2G; (c) 2A, 2B, 2C, 2J, and 2M; (d) 2A, 2B, 2C, 2D, 2E, and2M; (e) 2A, 2B, 2C, 2K, and 2G; (f) 2A, 2B, 2C, 2D, 2L, and 2G; (g) 2A,2I, 2D, 2E, 2F, and 2G; (h) 2A, 2I, 2D, 2E, and 2M; (i) 2A, 2I, 2J, 2F,and 2G; (j) 2A, 2I, 2J, and 2M; (k) 2A, 2I, 2K, and 2G; (l) 2A, 2I, 2D,2L and 2G; (m) 2H, 2C, 2D, 2E, 2F, and 2G; (n) 2H, 2C, 2J, 2F, and 2G;(o) 2H, 2C, 2J, and 2M; (p) 2H, 2C, 2D, 2E, and 2M; (q) 2H, 2C, 2K, and2G; or (r) 2H, 2C, 2D, 2L, and 2G. In some embodiments, 1M isspontaneous. In other embodiments, 1M is an EM12. In one embodiment, 2Jis an EB11. In another embodiment, 2J is an EB12.

In one embodiment, the NNOMO comprises (1) a MMP comprising 1A and 1B;1J; 1J and 1K; 1A, 1B, 1C, 1D, and 1E; 1A, 1B, 1C, 1D and 1F; 1J, 1C, 1Dand 1E; 1J, 1C, 1D and 1F; 1J and 1L; 1J, 1M, 1N and 1O; 1J, 1N and 1O;1J, 1K, 1C, 1D and 1E; 1J, 1K, 1C, 1D and 1F; 1I; 1A, 1B, 1C, 1D, 1E and1I; 1A, 1B, 1C, 1D, 1F and 1I; 1J, 1C, 1D, 1E and 1I; 1J, 1C, 1D, 1F and1I; 1J, 1L and 1I; 1J, 1M, 1N, 1O and 1I; 1J, 1N, 1O and 1I; 1J, 1K, 1C,1D, 1E and 1I; 1J, 1K, 1C, 1D, 1F and 1I; 1G; 1A, 1B, 1C, 1D, 1E and 1G;1A, 1B, 1C, 1D, 1F and 1G; 1J, 1C, 1D, 1E and 1G; 1J, 1C, 1D, 1F and 1G;1J, 1L and 1G; 1J, 1M, 1N, 1O and 1G; 1J, 1N, 1O and 1G; 1J, 1K, 1C, 1D,1E and 1G; 1J, 1K, 1C, 1D, 1F and 1G; 1G and 1H; 1A, 1B, 1C, 1D, 1E, 1Gand 1H; 1A, 1B, 1C, 1D, 1F, 1G and 1H; 1J, 1C, 1D, 1E, 1G and 1H; 1J,1C, 1D, 1F, 1G and 1H; 1J, 1L, 1G and 1H; 1J, 1M, 1N, 1O, 1G and 1H; 1J,1N, 1O, 1G and 1H; 1J, 1K, 1C, 1D, 1E, 1G and 1H; or 1J, 1K, 1C, 1D, 1F,1G and 1H; and (2) a BDOP. In some embodiments, 1K is spontaneous. Inother embodiments, 1K is an EM10. In some embodiments, 1M is an EM12.

Any MMP provided herein can be combined with any BDOP provided herein.

Also provided herein are exemplary pathways, which utilize formaldehydeproduced from the oxidation of methanol (e.g., as provided in FIG. 1,step J) in the formation of intermediates of certain central metabolicpathways that can be used for the formation of biomass. One exemplaryFAP that can utilize formaldehyde produced from the oxidation ofmethanol (e.g., as provided in FIG. 1) is shown in FIG. 3, whichinvolves condensation of formaldehyde and D-ribulose-5-phosphate to formH6P by EF1 (FIG. 3, step A). The enzyme can use Mg²⁺ or Mn²⁺ for maximalactivity, although other metal ions are useful, and evennon-metal-ion-dependent mechanisms are contemplated. H6P is convertedinto F6P by EF2 (FIG. 3, step B). Another exemplary pathway thatinvolves the detoxification and assimilation of formaldehyde producedfrom the oxidation of methanol (e.g., as provided in FIG. 1) is shown inFIG. 4 and proceeds through DHA. EF3 is a special transketolase thatfirst transfers a glycoaldehyde group from xylulose-5-phosphate toformaldehyde, resulting in the formation of DHA and G3P, which is anintermediate in glycolysis (FIG. 4, step A). The DHA obtained from DHAsynthase is then further phosphorylated to form DHA phosphate by a DHAkinase (FIG. 4, step B). DHAP can be assimilated into glycolysis andseveral other pathways. Rather than converting formaldehyde to formateand on to CO₂ off-gassed, the pathways provided in FIGS. 3 and 4 showthat carbon is assimilated, going into the final product.

Thus, in one embodiment, an organism having a MMP, either alone or incombination with a BDOP, as provided herein, further comprises a FAPthat utilizes formaldehyde, e.g., obtained from the oxidation ofmethanol, in the formation of intermediates of certain central metabolicpathways that can be used, for example, in the formation of biomass. Insome embodiments, the FAP comprises 3A or 3B, wherein 3A is an EF1 and3B is an EF2 In other embodiments, the FAP comprises 4A or 4B, wherein4A is an EF3 and 4B is an EF4.

In certain embodiments, provided herein is a NNOMO having a MMP, whereinsaid organism comprises at least one exogenous nucleic acid encoding anEM9 (1J) expressed in a sufficient amount to enhance the availability ofreducing equivalents in the presence of methanol and/or expressed in asufficient amount to convert methanol to formaldehyde. In someembodiments, the organism comprises at least one exogenous nucleic acidencoding an EM9 expressed in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol. Inother embodiments, the organism comprises at least one exogenous nucleicacid encoding an EM9 expressed in a sufficient amount to convertmethanol to formaldehyde. In some embodiments, the microbial organismfurther comprises a FAP. In certain embodiments, the organism furthercomprises at least one exogenous nucleic acid encoding a FAPE expressedin a sufficient amount to produce an intermediate of glycolysis and/or ametabolic pathway that can be used, for example, in the formation ofbiomass. In certain embodiments, the FAPE is selected from the groupconsisting of an EF1 (3A), an EF2 (3B), an EF3 (4A) and an EF4 (4B).

In some embodiments, the exogenous nucleic acid encoding an EM9 isexpressed in a sufficient amount to produce an amount of formaldehydegreater than or equal to 1 μM, 10 μM, 20 μM, or 50 μM, or a rangethereof, in culture medium or intracellularly. In other embodiments, theexogenous nucleic acid encoding an EM9 is capable of producing an amountof formaldehyde greater than or equal to 1 μM, 10 μM, 20 μM, or 50 μM,or a range thereof, in culture medium or intracellularly. In someembodiments, the range is from 1 μM to 50 μM or greater. In otherembodiments, the range is from 10 μM to 50 μM or greater. In otherembodiments, the range is from 20 μM to 50 μM or greater. In otherembodiments, the amount of formaldehyde production is 50 μM or greater.In specific embodiments, the amount of formaldehyde production is inexcess of, or as compared to, that of a negative control, e.g., the samespecies of organism that does not comprise the exogenous nucleic acid,such as a wild-type microbial organism or a control microbial organismthereof. In certain embodiments, the EM9 is selected from those providedherein, e.g., as exemplified in Example I (see FIG. 1, step J). Incertain embodiments, the amount of formaldehyde production is determinedby a whole cell assay, such as that provided in Example I (see FIG. 1,step J), or by another assay provided herein or otherwise known in theart. In certain embodiments, formaldehyde utilization activity is absentin the whole cell.

In certain embodiments, the exogenous nucleic acid encoding an EM9 isexpressed in a sufficient amount to produce at least 1×, 2×, 3×, 4×, 5×,6×, 7×, 8×, 9×, 10×, 15×, 20×, 30×, 40×, 50×, 100× or more formaldehydein culture medium or intracellularly. In other embodiments, theexogenous nucleic acid encoding an EM9 is capable of producing an amountof formaldehyde at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×,20×, 30×, 40×, 50×, 100×, or a range thereof, in culture medium orintracellularly. In some embodiments, the range is from 1× to 100×. Inother embodiments, the range is from 2× to 100×. In other embodiments,the range is from 5× to 100×. In other embodiments, the range is from10× to 100×. In other embodiments, the range is from 50× to 100×. Insome embodiments, the amount of formaldehyde production is at least 20×.In other embodiments, the amount of formaldehyde production is at least50×. In specific embodiments, the amount of formaldehyde production isin excess of, or as compared to, that of a negative control, e.g., thesame species of organism that does not comprise the exogenous nucleicacid, such as a wild-type microbial organism or a control microbialorganism thereof. In certain embodiments, the EM9 is selected from thoseprovided herein, e.g., as exemplified in Example I (see FIG. 1, step J).In certain embodiments, the amount of formaldehyde production isdetermined by a whole cell assay, such as that provided in Example I(see FIG. 1, step J), or by another assay provided herein or otherwiseknown in the art. In certain embodiments, formaldehyde utilizationactivity is absent in the whole cell.

In one aspect, provided herein is a NNOMO, comprising (1) a MMP, whereinsaid organism comprises at least one exogenous nucleic acid encoding aMMPE in a sufficient amount to enhance the availability of reducingequivalents in the presence of methanol and/or expressed in a sufficientamount to convert methanol to formaldehyde; and (2) a FAP, wherein saidorganism comprises at least one exogenous nucleic acid encoding a FAPEexpressed in a sufficient amount to produce an intermediate ofglycolysis and/or a metabolic pathway that can be used, for example, inthe formation of biomass. In some embodiments, the organism comprises atleast one exogenous nucleic acid encoding an EM9 expressed in asufficient amount to enhance the availability of reducing equivalents inthe presence of methanol. In other embodiments, the organism comprisesat least one exogenous nucleic acid encoding an EM9 expressed in asufficient amount to convert methanol to formaldehyde. In specificembodiments, the MMP comprises an EM9 (1J). In certain embodiments, theFAPE is 3A, and the intermediate is a H6P, a F6P, or a combinationthereof. In other embodiments, the FAPE is 3B, and the intermediate is aH6P, a F6P, or a combination thereof. In yet other embodiments, the FAPEis 3A and 3B, and the intermediate is a H6P, a F6P, or a combinationthereof. In some embodiments, the FAPE is 4A, and the intermediate is aDHA, a DHA phosphate, or a combination thereof. In other embodiments,the FAPE is 4B, and the intermediate is a DHA, a DHA phosphate, or acombination thereof. In yet other embodiments, the FAPE is 4A and 4B,and the intermediate is a DHA, a DHA phosphate, or a combinationthereof. In one embodiment, the at least one exogenous nucleic acidencoding the MMPE, in the presence of methanol, sufficiently enhancesthe availability of reducing equivalents and sufficiently increasesformaldehyde assimilation to increase the production of BDO or otherproducts described herein by the non-naturally microbial organism. Insome embodiments, the MMP comprises any of the various combinations ofMMPEs described above or elsewhere herein.

In certain embodiments, (1) the MMP comprises: 1A, 1B, 1C, 1D, 1E, 1F,1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or any combination of 1A, 1B, 1C,1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O, thereof, wherein 1Ais an EM1; 1B is an EM2; 1C is an EM3; 1D is an EM4; 1E is an EM5; 1F isan EM6; 1G is an EM15; 1H is an EM16, 1I is an EM8; 1J is an EM9; 1K isspontaneous or an EM1O; 1L is an EM11; 1M is spontaneous or an EM12; 1Nis an EM13 and 1O is EM14; and (2) the FAP comprises 3A, 3B or acombination thereof, wherein 3A is an EF1, and 3B is an EF2. In someembodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. Insome embodiments, 1M is spontaneous. In other embodiments, 1M is anEM12. In some embodiments, the intermediate is a H6P. In otherembodiments, the intermediate is a F6P. In yet other embodiments, theintermediate is a H6P and a F6P.

In one embodiment, the FAP comprises 3A. In another embodiment, the FAPcomprises 3B. In one embodiment, the FAP comprises 3A and 3B.

In some embodiments, the MMP is a MMP depicted in FIG. 1, and a FAPdepicted in FIG. 3. An exemplary set of FAPEs to convertD-ribulose-5-phosphate and formaldehyde to F6P (via H6P) according toFIG. 3 include 3A and 3B.

In a specific embodiment, (1) the MMP comprises 1J; and (2) the FAPcomprises 3A and 3B. In other embodiments, (1) the MMP comprises 1J and1K; and (2) the FAP comprises 3A and 3B. In some embodiments, (1) theMMP comprises 1J, 1C, 1D and 1E; and (2) the FAP comprises 3A and 3B. Inone embodiment, (1) the MMP comprises 1J, 1C, 1D and 1F; and (2) the FAPcomprises 3A and 3B. In another embodiment, (1) the MMP comprises 1J and1L; and (2) the FAP comprises 3A and 3B. In yet another embodiment, (1)the MMP comprises 1J, 1M, 1N and 1O; and (2) the FAP comprises 3A and3B. In certain embodiments, (1) the MMP comprises 1J, 1N and 1O; and (2)the FAP comprises 3A and 3B. In some embodiments, (1) the MMP comprises1J, 1K, 1C, 1D and 1E; and (2) the FAP comprises 3A and 3B. In oneembodiment, (1) the MMP comprises 1J, 1K, 1C, 1D and 1F; and (2) the FAPcomprises 3A and 3B. In some embodiments, (1) the MMP comprises 1J, 1C,1D, 1E and 1I; and (2) the FAP comprises 3A and 3B. In one embodiment,(1) the MMP comprises 1J, 1C, 1D, 1F and 1I; and (2) the FAP comprises3A and 3B. In another embodiment, (1) the MMP comprises 1J, 1L and 1I;and (2) the FAP comprises 3A and 3B. In yet another embodiment, (1) theMMP comprises 1J, 1M, 1N, 1O and 1I; and (2) the FAP comprises 3A and3B. In certain embodiments, (1) the MMP comprises 1J, 1N, 1O and 1I; and(2) the FAP comprises 3A and 3B. In some embodiments, (1) the MMPcomprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the FAP comprises 3A and3B. In one embodiment, (1) the MMP comprises 1J, 1K, 1C, 1D, 1F and 1I;and (2) the FAP comprises 3A and 3B. In some embodiments, (1) the MMPcomprises 1J, 1C, 1D, 1E and 1G; and (2) the FAP comprises 3A and 3B. Inone embodiment, (1) the MMP comprises 1J, 1C, 1D, 1F and 1G; and (2) theFAP comprises 3A and 3B. In another embodiment, (1) the MMP comprises1J, 1L and 1G; and (2) the FAP comprises 3A and 3B. In yet anotherembodiment, (1) the MMP comprises 1J, 1M, 1N, 1O and 1G; and (2) the FAPcomprises 3A and 3B. In certain embodiments, (1) the MMP comprises 1J,1N, 1O and 1G; and (2) the FAP comprises 3A and 3B. In some embodiments,(1) the MMP comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the FAPcomprises 3A and 3B. In one embodiment, (1) the MMP comprises 1J, 1K,1C, 1D, 1F and 1G; and (2) the FAP comprises 3A and 3B. In someembodiments, (1) the MMP comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2)the FAP comprises 3A and 3B. In one embodiment, (1) the MMP comprises1J, 1C, 1D, 1F, 1G and 1H; and (2) the FAP comprises 3A and 3B. Inanother embodiment, (1) the MMP comprises 1J, 1L, 1G and 1H; and (2) theFAP comprises 3A and 3B. In yet another embodiment, (1) the MMPcomprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the FAP comprises 3A and3B. In certain embodiments, (1) the MMP comprises 1J, 1N, 1O, 1G and 1H;and (2) the FAP comprises 3A and 3B. In some embodiments, (1) the MMPcomprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the FAP comprises 3Aand 3B. In one embodiment, (1) the MMP comprises 1J, 1K, 1C, 1D, 1F, 1Gand 1H; and (2) the FAP comprises 3A and 3B. In some embodiments, 1K isspontaneous. In other embodiments, 1K is an EM10. In some embodiments,1M is spontaneous. In some embodiments, the intermediate is a H6P. Inother embodiments, the intermediate is a F6P. In yet other embodiments,the intermediate is a H6P and a F6P.

In certain embodiments, (1) the MMP comprises: 1A, 1B, 1C, 1D, 1E, 1F,1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 10 or any combination of 1A, 1B, 1C,1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O, thereof, wherein 1Ais an EM1; 1B is an EM2; 1C is an EM3; 1D is an EM4; 1E is an EM5; 1F isan EM6; 1G is an EM15; 1H is an EM16, 1I is an EM8; 1J is an EM9; 1K isspontaneous or an EM1O; 1L is an EM11; 1M is spontaneous or an EM12; 1Nis an EM13 and 1O is EM14; and (2) the FAP comprises 4A, 4B or acombination thereof, wherein 4A is an EF3 and 4B is an EF4. In someembodiments, 1K is spontaneous. In other embodiments, 1K is an EM10. Insome embodiments, 1M is spontaneous. In other embodiments, 1M is anEM12. In some embodiments, the intermediate is a DHA. In otherembodiments, the intermediate is a DHA phosphate. In yet otherembodiments, the intermediate is a DHA and a DHA phosphate.

In one embodiment, the FAP comprises 4A. In another embodiment, the FAPcomprises 4B. In one embodiment, the FAP comprises 4A and 4B.

In some embodiments, the MMP is a MMP depicted in FIG. 1, and a FAPdepicted in FIG. 4. An exemplary set of FAPEs to convertxyulose-5-phosphate and formaldehyde to DHA-phosphate (via DHA)according to FIG. 4 include 4A and 4B.

In a specific embodiment, (1) the MMP comprises 1J; and (2) the FAPcomprises 4A and 4B. In other embodiments, (1) the MMP comprises 1J and1K; and (2) the FAP comprises 4A and 4B. In some embodiments, (1) theMMP comprises 1J, 1C, 1D and 1E; and (2) the FAP comprises 4A and 4B. Inone embodiment, (1) the MMP comprises 1J, 1C, 1D and 1F; and (2) the FAPcomprises 4A and 4B. In another embodiment, (1) the MMP comprises 1J and1L; and (2) the FAP comprises 4A and 4B. In yet another embodiment, (1)the MMP comprises 1J, 1M, 1N and 1O; and (2) the FAP comprises 4A and4B. In certain embodiments, (1) the MMP comprises 1J, 1N and 1O; and (2)the FAP comprises 4A and 4B. In some embodiments, (1) the MMP comprises1J, 1K, 1C, 1D and 1E; and (2) the FAP comprises 4A and 4B. In oneembodiment, (1) the MMP comprises 1J, 1K, 1C, 1D and 1F; and (2) the FAPcomprises 4A and 4B. In some embodiments, (1) the MMP comprises 1J, 1C,1D, 1E and 1I; and (2) the FAP comprises 4A and 4B. In one embodiment,(1) the MMP comprises 1J, 1C, 1D, 1F and 1I; and (2) the FAP comprises4A and 4B. In another embodiment, (1) the MMP comprises 1J, 1L and 1I;and (2) the FAP comprises 4A and 4B. In yet another embodiment, (1) theMMP comprises 1J, 1M, 1N, 1O and 1I; and (2) the FAP comprises 4A and4B. In certain embodiments, (1) the MMP comprises 1J, 1N, 1O and 1I; and(2) the FAP comprises 4A and 4B. In some embodiments, (1) the MMPcomprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the FAP comprises 4A and4B. In one embodiment, (1) the MMP comprises 1J, 1K, 1C, 1D, 1F and 1I;and (2) the FAP comprises 4A and 4B. In some embodiments, (1) the MMPcomprises 1J, 1C, 1D, 1E and 1G; and (2) the FAP comprises 4A and 4B. Inone embodiment, (1) the MMP comprises 1J, 1C, 1D, 1F and 1G; and (2) theFAP comprises 4A and 4B. In another embodiment, (1) the MMP comprises1J, 1L and 1G; and (2) the FAP comprises 4A and 4B. In yet anotherembodiment, (1) the MMP comprises 1J, 1M, 1N, 1O and 1G; and (2) the FAPcomprises 4A and 4B. In certain embodiments, (1) the MMP comprises 1J,1N, 1O and 1G; and (2) the FAP comprises 4A and 4B. In some embodiments,(1) the MMP comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the FAPcomprises 4A and 4B. In one embodiment, (1) the MMP comprises 1J, 1K,1C, 1D, 1F and 1G; and (2) the FAP comprises 4A and 4B. In someembodiments, (1) the MMP comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2)the FAP comprises 4A and 4B. In one embodiment, (1) the MMP comprises1J, 1C, 1D, 1F, 1G and 1H; and (2) the FAP comprises 4A and 4B. Inanother embodiment, (1) the MMP comprises 1J, 1L, 1G and 1H; and (2) theFAP comprises 4A and 4B. In yet another embodiment, (1) the MMPcomprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the FAP comprises 4A and4B. In certain embodiments, (1) the MMP comprises 1J, 1N, 1O, 1G and 1H;and (2) the FAP comprises 4A and 4B. In some embodiments, (1) the MMPcomprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the FAP comprises 4Aand 4B. In one embodiment, (1) the MMP comprises 1J, 1K, 1C, 1D, 1F, 1Gand 1H; and (2) the FAP comprises 4A and 4B. In some embodiments, 1K isspontaneous. In other embodiments, 1K is an EM10. In some embodiments,1M is spontaneous. In some embodiments, the intermediate is a DHA. Inother embodiments, the intermediate is a DHA phosphate. In yet otherembodiments, the intermediate is a DHA and a DHA phosphate.

Any MMP provided herein can be combined with any FAP provided herein. Inaddition, any MMP provided herein can be combined with any BDOP and anyformaldehyde pathway provided herein.

Also provided herein are methods of producing formaldehyde comprisingculturing a NNOMO having a MMP provided herein. In some embodiments, theMMP comprises 1J. In certain embodiments, the organism is cultured in asubstantially anaerobic culture medium. In specific embodiments, theformaldehyde is an intermediate that is consumed (assimilated) in theproduction of BDO and other products described herein.

Also provided herein are methods of producing an intermediate ofglycolysis and/or a metabolic pathway that can be used, for example, inthe formation of biomass, comprising culturing a NNOMO having a MMP anda FAP, as provided herein, under conditions and for a sufficient periodof time to produce the intermediate. In some embodiments, theintermediate is a H6P. In other embodiments, the intermediate is a F6P.In yet other embodiments, the intermediate is a H6P and a F6P. In someembodiments, the intermediate is a DHA. In other embodiments, theintermediate is a DHA phosphate. In yet other embodiments, theintermediate is a DHA and a DHA phosphate. In some embodiments, the MMPcomprises 1J. In certain embodiments, the organism is cultured in asubstantially anaerobic culture medium. Such biomass can also be used inmethods of producing any of the products, such as the biobased products,provided elsewhere herein.

In some embodiments, the organism comprises two, three, four, five, six,seven, eight or more exogenous nucleic acids, each encoding a BDOPE. Insome embodiments, the organism comprises two exogenous nucleic acids,each encoding a BDOPE. In some embodiments, the organism comprises threeexogenous nucleic acids, each encoding a BDOPE. In some embodiments, theorganism comprises four exogenous nucleic acids, each encoding a BDOPE.In other embodiments, the organism comprises five exogenous nucleicacids, each encoding a BDOPE. In some embodiments, the organismcomprises six exogenous nucleic acids, each encoding a BDOPE. In otherembodiments, the organism comprises seven exogenous nucleic acids, eachencoding a BDOPE. In certain embodiments, the organism comprises two,three, four, five, six or seven exogenous nucleic acids, each encoding aBDOPE; and the organism further comprises two, three, four, five, six orseven exogenous nucleic acids, each encoding a MMPE. In certainembodiments, the organism further comprises two exogenous nucleic acids,each encoding a MMPE. In certain embodiments, the organism furthercomprises three exogenous nucleic acids, each encoding a MMPE. Incertain embodiments, the organism comprises further four exogenousnucleic acids, each encoding a MMPE. In certain embodiments, theorganism further comprises five exogenous nucleic acids, each encoding aMMPE. In certain embodiments, the organism further comprises sixexogenous nucleic acids, each encoding a MMPE. In certain embodiments,the organism further comprises seven exogenous nucleic acids, eachencoding a MMPE.

In some embodiments, the organism comprises two or more exogenousnucleic acids, each encoding a FAPE. In some embodiments, the organismcomprises two exogenous nucleic acids, each encoding a FAPE. In certainembodiments, the organism comprises two exogenous nucleic acids, eachencoding a FAPE; and the organism further comprises two, three, four,five, six or seven exogenous nucleic acids, each encoding a MMPE. Incertain embodiments, the organism further comprises two exogenousnucleic acids, each encoding a MMPE. In certain embodiments, theorganism further comprises three exogenous nucleic acids, each encodinga MMPE. In certain embodiments, the organism comprises further fourexogenous nucleic acids, each encoding a MMPE. In certain embodiments,the organism further comprises five exogenous nucleic acids, eachencoding a MMPE. In certain embodiments, the organism further comprisessix exogenous nucleic acids, each encoding a MMPE. In certainembodiments, the organism further comprises seven exogenous nucleicacids, each encoding a MMPE.

In some embodiments, the at least one exogenous nucleic acid encoding aMMPE is a heterologous nucleic acid. In other embodiments, the at leastone exogenous nucleic acid encoding a BDOPE is a heterologous nucleicacid. In other embodiments, the at least one exogenous nucleic acidencoding a FAPE is a heterologous nucleic acid. In certain embodiments,the at least one exogenous nucleic acid encoding a MMPE is aheterologous nucleic acid, and the at least one exogenous nucleic acidencoding a BDOPE is a heterologous nucleic acid. In other embodiments,the at least one exogenous nucleic acid encoding a MMPE is aheterologous nucleic acid, and the at least one exogenous nucleic acidencoding a FAPE is a heterologous nucleic acid.

In certain embodiments, the organism is in a substantially anaerobicculture medium.

In some embodiments, formaldehyde produced from EM9 (FIG. 1, step J) incertain of the NNOMO provided herein is used for generating energy,redox and/or formation of biomass. Two such pathways are shown in FIG.3. Additionally, several organisms use an alternative pathway called the“serine cycle” for formaldehyde assimilation. These organisms includethe methylotroph, Methylobacterium extorquens AM1, and another,Methylobacterium organophilum. The net balance of this cycle is thefixation of two mols of formaldehyde and 1 mol of CO₂ into 1 mol of3-phosphoglycerate, which is used for biosynthesis, at the expense of 2mols ATP and the oxidation of 2 mols of NAD(P)H.

In the first reaction of the serine pathway, formaldehyde reacts withglycine to form serine. The reaction is catalyzed by serinehydroxymethyltransferase (SHMT), an enzyme that uses tetrahydrofolate(THF) as a cofactor. This leads to the formation of5,10-methylenetetrahydrofolate. During the reaction, formaldehyde istransferred from 5,10-methylenetetrahydrofolate to the glycine, formingL-serine. In the next step, serine is transaminated with glyoxylate asthe amino group acceptor by the enzyme serine-glyoxylateaminotransferase, to produce hydroxypyruvate and glycine.Hydroxypyruvate is reduced to glycerate by hydroxypyruvate reductase.Glycerate 2-kinase catalyzes the addition of a phosphate group from ATPto produce 2-phosphoglycerate.

Some of the 2-phosphoglycerate is converted by phosphoglycerate mutaseto 3-phosphoglycerate, which is an intermediate of the central metabolicpathways and used for biosynthesis. The rest of the 2-phosphoglycerateis converted by an enolase to phosphoenolpyruvate (PEP). PEP carboxylasethen catalyzes the fixation of carbon dioxide coupled to the conversionof PEP to oxaloacetate, which is reduced to malate by malatedehydrogenase, an NAD-linked enzyme. Malate is activated to malylcoenzyme A by malate thiokinase and is cleaved by malyl coenzyme A lyaseinto acetyl coA and glyoxylate. These two enzymes (malate thiokinase andmalyl coenzyme A lyase), as well as hydroxypyruvate reductase andglycerate-2-kinase, are uniquely present in methylotrophs that containthe serine pathway.

In organisms that possess isocitrate lyase, a key enzyme of theglyoxylate cycle, acetyl CoA is converted to glyoxylate by theglyoxylate cycle. However, if the enzyme is missing, it is converted byanother unknown pathway (deVries et al, FEMS Microbiol Rev, 6 (1):57-101 (1990)). The resulting glyoxylate can serve as substrate forserine-glyoxylate aminotransferase, regenerating glycine and closing thecircle.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the figures, including the pathwaysof FIGS. 1, 2, 3 and 4 can be utilized to generate a NNOMO that producesany pathway intermediate or product, as desired. Non-limiting examplesof such intermediate or products are 4-HB and BDO. As disclosed herein,such a microbial organism that produces an intermediate can be used incombination with another microbial organism expressing downstreampathway enzymes to produce a desired product. However, it is understoodthat a non-naturally occurring organism that produces a BDOPintermediate can be utilized to produce the intermediate as a desiredproduct (e.g., 4-hydroxybutanal).

In certain embodiments, a NNOMO comprising a MMP and a BDOP providedherein, further comprises one or more gene disruptions. In certainembodiments, the one or more gene disruptions confer increasedproduction of BDO in the organism. In other embodiments, a NNOMOcomprising a MMP and a FAP provided herein, further comprises one ormore gene disruptions. In some embodiments, the gene disruption is in anendogenous gene encoding a protein and/or enzyme involved in nativeproduction of ethanol, glycerol, acetate, lactate, formate, CO₂, aminoacids, or any combination thereof, by said microbial organism. In oneembodiment, the gene disruption is in an endogenous gene encoding aprotein and/or enzyme involved in native production of ethanol. Inanother embodiment, the gene disruption is in an endogenous geneencoding a protein and/or enzyme involved in native production ofglycerol. In other embodiments, the gene disruption is in an endogenousgene encoding a protein and/or enzyme involved in native production ofacetate. In another embodiment, the gene disruption is in an endogenousgene encoding a protein and/or enzyme involved in native production oflactate. In one embodiment, the gene disruption is in an endogenous geneencoding a protein and/or enzyme involved in native production offormate. In another embodiment, the gene disruption is in an endogenousgene encoding a protein and/or enzyme involved in native production ofCO₂. In other embodiments, the gene disruption is in an endogenous geneencoding a protein and/or enzyme involved in native production of aminoacids by said microbial organism. In some embodiments, the protein orenzyme is a pyruvate decarboxylase, an ethanol dehydrogenase, a glyceroldehydrogenase, a glycerol-3-phosphatase, a glycerol-3-phosphatedehydrogenase, a lactate dehydrogenase, an acetate kinase, aphosphotransacetylase, a pyruvate oxidase, a pyruvate:quinoneoxidoreductase, a pyruvate formate lyase, an alcohol dehydrogenase, alactate dehydrogenase, a pyruvate dehydrogenase, a pyruvateformate-lyase-2-iol. 175:377-385 (1993))se, a pyruvate transporter, amonocarboxylate transporter, a NADH dehydrogenase, a cytochrome oxidase,a pyruvate kinase, or any combination thereof. In certain embodiments,the one or more gene disruptions confer increased production offormaldehyde in the organism. In another embodiment, the gene disruptionis in an endogenous gene encoding a protein and/or enzyme involved in anative formaldehyde utilization pathway. In certain embodiments, theorganism comprises from one to twenty-five gene disruptions. In otherembodiments, the organism comprises from one to twenty gene disruptions.In some embodiments, the organism comprises from one to fifteen genedisruptions. In other embodiments, the organism comprises from one toten gene disruptions. In some embodiments, the organism comprises fromone to five gene disruptions. In certain embodiments, the organismcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25 gene disruptions or more.

In other embodiments, a NNOMO comprising a MMP and a BDOP providedherein, further comprises one or more endogenous proteins or enzymesinvolved in native production of ethanol, glycerol, acetate, lactate,formate, CO₂ and/or amino acids by said microbial organism, wherein saidone or more endogenous proteins or enzymes has attenuated protein orenzyme activity and/or expression levels. In some embodiments, a NNOMOcomprising a MMP and a FAP provided herein, further comprises one ormore endogenous proteins or enzymes involved in native production ofethanol, glycerol, acetate, lactate, formate, CO₂ and/or amino acids bysaid microbial organism, wherein said one or more endogenous proteins orenzymes has attenuated protein or enzyme activity and/or expressionlevels. In one embodiment the endogenous protein or enzyme is a pyruvatedecarboxylase, an ethanol dehydrogenase, a glycerol dehydrogenase, aglycerol-3-phosphatase, a glycerol-3-phosphate dehydrogenase, a lactatedehydrogenase, an acetate kinase, a phosphotransacetylase, a pyruvateoxidase, a pyruvate:quinone oxidoreductase, a pyruvate formate lyase, analcohol dehydrogenase, a lactate dehydrogenase, a pyruvatedehydrogenase, a pyruvate formate-lyase-2-ketobutyrate formate-lyase, apyruvate transporter, a monocarboxylate transporter, a NADHdehydrogenase, a cytochrome oxidase, a pyruvate kinase, or anycombination thereof.

Each of the non-naturally occurring alterations provided herein resultin increased production and an enhanced level of BDO, for example,during the exponential growth phase of the microbial organism, comparedto a strain that does not contain such metabolic alterations, underappropriate culture conditions. Appropriate conditions include, forexample, those disclosed herein, including conditions such as particularcarbon sources or reactant availabilities and/or adaptive evolution.

In certain embodiments, provided herein are NNOMO having geneticalterations such as gene disruptions that increase production of, forexample, BDO, for example, growth-coupled production of BDO. Productproduction can be, for example, obligatorily linked to the exponentialgrowth phase of the microorganism by genetically altering the metabolicpathways of the cell, as disclosed herein. The genetic alterations canincrease the production of the desired product or even make the desiredproduct an obligatory product during the growth phase. Appropriateconditions include, for example, those disclosed herein, includingconditions such as particular carbon sources or reactant availabilitiesand/or adaptive evolution.

Given the teachings and guidance provided herein, those skilled in theart will understand that to introduce a metabolic alteration such asattenuation of an enzyme, it can be necessary to disrupt the catalyticactivity of the one or more enzymes involved in the reaction.Alternatively, a metabolic alteration can include disrupting expressionof a regulatory protein or cofactor necessary for enzyme activity ormaximal activity. Furthermore, genetic loss of a cofactor necessary foran enzymatic reaction can also have the same effect as a disruption ofthe gene encoding the enzyme. Disruption can occur by a variety ofmethods including, for example, deletion of an encoding gene orincorporation of a genetic alteration in one or more of the encodinggene sequences. The encoding genes targeted for disruption can be one,some, or all of the genes encoding enzymes involved in the catalyticactivity. For example, where a single enzyme is involved in a targetedcatalytic activity, disruption can occur by a genetic alteration thatreduces or eliminates the catalytic activity of the encoded geneproduct. Similarly, where the single enzyme is multimeric, includingheteromeric, disruption can occur by a genetic alteration that reducesor destroys the function of one or all subunits of the encoded geneproducts. Destruction of activity can be accomplished by loss of thebinding activity of one or more subunits required to form an activecomplex, by destruction of the catalytic subunit of the multimericcomplex or by both. Other functions of multimeric protein associationand activity also can be targeted in order to disrupt a metabolicreaction. Such other functions are well known to those skilled in theart. Similarly, a target enzyme activity can be reduced or eliminated bydisrupting expression of a protein or enzyme that modifies and/oractivates the target enzyme, for example, a molecule required to convertan apoenzyme to a holoenzyme. Further, some or all of the functions of asingle polypeptide or multimeric complex can be disrupted in order toreduce or abolish the catalytic activity of one or more enzymes involvedin a reaction or metabolic modification provided herein. Similarly, someor all of enzymes involved in a reaction or metabolic modificationprovided herein can be disrupted so long as the targeted reaction isreduced or eliminated.

Given the teachings and guidance provided herein, those skilled in theart also will understand that an enzymatic reaction can be disrupted byreducing or eliminating reactions encoded by a common gene and/or by oneor more orthologs of that gene exhibiting similar or substantially thesame activity. Reduction of both the common gene and all orthologs canlead to complete abolishment of any catalytic activity of a targetedreaction. However, disruption of either the common gene or one or moreorthologs can lead to a reduction in the catalytic activity of thetargeted reaction sufficient to promote coupling of growth to productbiosynthesis. Exemplified herein are both the common genes encodingcatalytic activities for a variety of metabolic modifications as well astheir orthologs. Those skilled in the art will understand thatdisruption of some or all of the genes encoding a enzyme of a targetedmetabolic reaction can be practiced in the methods provided herein andincorporated into the NNOMO in order to achieve the increased productionof BDO or growth-coupled product production.

Given the teachings and guidance provided herein, those skilled in theart also will understand that enzymatic activity or expression can beattenuated using well known methods. Reduction of the activity or amountof an enzyme can mimic complete disruption of a gene if the reductioncauses activity of the enzyme to fall below a critical level that isnormally required for a pathway to function. Reduction of enzymaticactivity by various techniques rather than use of a gene disruption canbe important for an organism's viability. Methods of reducing enzymaticactivity that result in similar or identical effects of a genedisruption include, but are not limited to: reducing gene transcriptionor translation; destabilizing mRNA, protein or catalytic RNA; andmutating a gene that affects enzyme activity or kinetics (See, Sambrooket al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold SpringHarbor Laboratory, New York (2001); and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1999). Natural or imposed regulatory controls can also accomplishenzyme attenuation including: promoter replacement (See, Wang et al.,Mol. Biotechnol. 52(2):300-308 (2012)); loss or alteration oftranscription factors (Dietrick et al., Annu. Rev. Biochem. 79:563-590(2010); and Simicevic et al., Mol. Biosyst. 6(3):462-468 (2010));introduction of inhibitory RNAs or peptides such as siRNA, antisenseRNA, RNA or peptide/small-molecule binding aptamers, ribozymes,aptazymes and riboswitches (Wieland et al., Methods 56(3):351-357(2012); O'Sullivan, Anal. Bioanal. Chem. 372(1):44-48 (2002); and Lee etal., Curr. Opin. Biotechnol. 14(5):505-511 (2003)); and addition ofdrugs or other chemicals that reduce or disrupt enzymatic activity suchas an enzyme inhibitor, an antibiotic or a target-specific drug.

One skilled in the art will also understand and recognize thatattenuation of an enzyme can be done at various levels. For example, atthe gene level, a mutation causing a partial or complete null phenotype,such as a gene disruption, or a mutation causing epistatic geneticeffects that mask the activity of a gene product (Miko, Nature Education1(1) (2008)), can be used to attenuate an enzyme. At the gene expressionlevel, methods for attenuation include: coupling transcription to anendogenous or exogenous inducer, such as isopropylthio-β-galactoside(IPTG), then adding low amounts of inducer or no inducer during theproduction phase (Donovan et al., J. Ind. Microbiol. 16(3):145-154(1996); and Hansen et al., Curr. Microbiol. 36(6):341-347 (1998));introducing or modifying a positive or a negative regulator of a gene;modify histone acetylation/deacetylation in a eukaryotic chromosomalregion where a gene is integrated (Yang et al., Curr. Opin. Genet. Dev.13(2):143-153 (2003) and Kurdistani et al., Nat. Rev. Mol. Cell Biol.4(4):276-284 (2003)); introducing a transposition to disrupt a promoteror a regulatory gene (Bleykasten-Brosshans et al., C. R. Biol.33(8-9):679-686 (2011); and McCue et al., PLoS Genet. 8(2):e1002474(2012)); flipping the orientation of a transposable element or promoterregion so as to modulate gene expression of an adjacent gene (Wang etal., Genetics 120(4):875-885 (1988); Hayes, Annu. Rev. Genet. 37:3-29(2003); in a diploid organism, deleting one allele resulting in loss ofheterozygosity (Daigaku et al., Mutation Research/Fundamental andMolecular Mechanisms of Mutagenesis 600(1-2)177-183 (2006)); introducingnucleic acids that increase RNA degradation (Houseley et al., Cell,136(4):763-776 (2009); or in bacteria, for example, introduction of atransfer-messenger RNA (tmRNA) tag, which can lead to RNA degradationand ribosomal stalling (Sunohara et al., RNA 10(3):378-386 (2004); andSunohara et al., J. Biol. Chem. 279:15368-15375 (2004)). At thetranslational level, attenuation can include: introducing rare codons tolimit translation (Angov, Biotechnol. J. 6(6):650-659 (2011));introducing RNA interference molecules that block translation (Castel etal., Nat. Rev. Genet. 14(2):100-112 (2013); and Kawasaki et al., Curr.Opin. Mol. Ther. 7(2):125-131 (2005); modifying regions outside thecoding sequence, such as introducing secondary structure into anuntranslated region (UTR) to block translation or reduce efficiency oftranslation (Ringner et al., PLoS Comput. Biol. 1(7):e72 (2005)); addingRNAase sites for rapid transcript degradation (Pasquinelli, Nat. Rev.Genet. 13(4):271-282 (2012); and Arraiano et al., FEMS Microbiol. Rev.34(5):883-932 (2010); introducing antisense RNA oligomers or antisensetranscripts (Nashizawa et al., Front. Biosci. 17:938-958 (2012));introducing RNA or peptide aptamers, ribozymes, aptazymes, riboswitches(Wieland et al., Methods 56(3):351-357 (2012); O'Sullivan, Anal.Bioanal. Chem. 372(1):44-48 (2002); and Lee et al., Curr. Opin.Biotechnol. 14(5):505-511 (2003)); or introducing translationalregulatory elements involving RNA structure that can prevent or reducetranslation that can be controlled by the presence or absence of smallmolecules (Araujo et al., Comparative and Functional Genomics, ArticleID 475731, 8 pages (2012)). At the level of enzyme localization and/orlongevity, enzyme attenuation can include: adding a degradation tag forfaster protein turnover (Hochstrasser, Annual Rev. Genet. 30:405-439(1996); and Yuan et al., PLoS One 8(4):e62529 (2013)); or adding alocalization tag that results in the enzyme being secreted or localizedto a subcellular compartment in a eukaryotic cell, where the enzymewould not be able to react with its normal substrate (Nakai et al.Genomics 14(4):897-911 (1992); and Russell et al., J. Bact.189(21)7581-7585 (2007)). At the level of post-translational regulation,enzyme attenuation can include: increasing intracellular concentrationof known inhibitors; or modifying post-translational modified sites(Mann et al., Nature Biotech. 21:255-261 (2003)). At the level of enzymeactivity, enzyme attenuation can include: adding an endogenous or anexogenous inhibitor, such as an enzyme inhibitor, an antibiotic or atarget-specific drug, to reduce enzyme activity; limiting availabilityof essential cofactors, such as vitamin B12, for an enzyme that requiresthe cofactor; chelating a metal ion that is required for enzymeactivity; or introducing a dominant negative mutation. The applicabilityof a technique for attenuation described above can depend upon whether agiven host microbial organism is prokaryotic or eukaryotic, and it isunderstand that a determination of what is the appropriate technique fora given host can be readily made by one skilled in the art.

In some embodiments, microaerobic designs can be used based on thegrowth-coupled formation of the desired product. To examine this,production cones can be constructed for each strategy by firstmaximizing and, subsequently minimizing the product yields at differentrates of biomass formation feasible in the network. If the rightmostboundary of all possible phenotypes of the mutant network is a singlepoint, it implies that there is a unique optimum yield of the product atthe maximum biomass formation rate possible in the network. In othercases, the rightmost boundary of the feasible phenotypes is a verticalline, indicating that at the point of maximum biomass the network canmake any amount of the product in the calculated range, including thelowest amount at the bottommost point of the vertical line. Such designsare given a low priority.

The BDO-production strategies identified by the methods disclosed hereinsuch as the OptKnock framework are generally ranked on the basis oftheir (i) theoretical yields, and (ii) growth-coupled BDO formationcharacteristics.

Accordingly, also provided herein is a NNOMO having metabolicmodifications coupling BDO production to growth of the organism, wherethe metabolic modifications includes disruption of one or more genesselected from the genes encoding proteins and/or enzymes providedherein.

Each of the strains can be supplemented with additional deletions if itis determined that the strain designs do not sufficiently increase theproduction of BDO and/or couple the formation of the product withbiomass formation. Alternatively, some other enzymes not known topossess significant activity under the growth conditions can becomeactive due to adaptive evolution or random mutagenesis. Such activitiescan also be knocked out. However, gene deletions provided herein allowthe construction of strains exhibiting high-yield production of BDO,including growth-coupled production of BDO.

In another aspect, provided herein is a method for producing BDO,comprising culturing any one of the NNOMOs comprising a MMP and an BDOPprovided herein under conditions and for a sufficient period of time toproduce BDO. In certain embodiments, the organism is cultured in asubstantially anaerobic culture medium.

Provided herein are methods for producing BDO, comprising culturing anorganism provided herein under conditions and for a sufficient period oftime to produce BDO. In some embodiments, the method comprisesculturing, for a sufficient period of time to produce BDO, a NNOMO,comprising (1) a MMP, wherein said organism comprises at least oneexogenous nucleic acid encoding a MMPE in a sufficient amount to enhancethe availability of reducing equivalents in the presence of methanol;and (2) a BDOP, comprising at least one exogenous nucleic acid encodinga BDOPE expressed in a sufficient amount to produce BDO.

In certain embodiments of the methods provided herein, the organismfurther comprises at least one nucleic acid encoding a BDOPE expressedin a sufficient amount to produce BDO. In some embodiments, the nucleicacid is an exogenous nucleic acid. In other embodiments, the nucleicacid is an endogenous nucleic acid. In some embodiments, the organismfurther comprises one or more gene disruptions provided herein thatconfer increased production of BDO in the organism. In certainembodiments, the one or more gene disruptions occurs in an endogenousgene encoding a protein or enzyme involved in native production ofethanol, glycerol, acetate, lactate, formate, CO₂ and/or amino acids bysaid microbial organism. In other embodiments, the organism furthercomprises one or more endogenous proteins or enzymes involved in nativeproduction of ethanol, glycerol, acetate, lactate, formate, CO₂ and/oramino acids by said microbial organism, wherein said one or moreendogenous proteins or enzymes has attenuated protein or enzyme activityand/or expression levels. In certain embodiments, the organism is aCrabtree positive, eukaryotic organism, and the organism is cultured ina culture medium comprising glucose. In certain embodiments, theorganism comprises from one to twenty-five gene disruptions. In otherembodiments, the organism comprises from one to twenty gene disruptions.In some embodiments, the organism comprises from one to fifteen genedisruptions. In other embodiments, the organism comprises from one toten gene disruptions. In some embodiments, the organism comprises fromone to five gene disruptions. In certain embodiments, the organismcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25 gene disruptions or more.

In an additional embodiment, provided is a NNOMO having a BDOP, FAPand/or MMP, wherein the NNOMO comprises at least one exogenous nucleicacid encoding an enzyme or protein that converts a substrate to aproduct. By way of example, in FIG. 1, the substrate of 1J is methanol,and the product is formaldehyde; the substrate of 1L is formaldehyde,and the product is formate; and so forth. One skilled in the art willunderstand that these are merely exemplary and that any of thesubstrate-product pairs disclosed herein suitable to produce a desiredproduct and for which an appropriate activity is available for theconversion of the substrate to the product can be readily determined byone skilled in the art based on the teachings herein. Thus, providedherein are NNOMOs containing at least one exogenous nucleic acidencoding an enzyme or protein, where the enzyme or protein converts thesubstrates and products of a MMP, such as that shown in FIG. 1; a BDOP,such as that shown in FIG. 2; and/or a FAP, such as that shown in FIG. 3or 4.

While generally described herein as a microbial organism that contains aBDOP, FAP and/or a MMP, it is understood that provided herein are alsoNNOMO comprising at least one exogenous nucleic acid encoding a BDO,formaldehyde assimilation and/or a MMPE expressed in a sufficient amountto produce an intermediate of a BDOP, FAP and/or a MMP intermediate. Forexample, as disclosed herein, a BDOP is exemplified in FIG. 2.Therefore, in addition to a microbial organism containing a BDOP thatproduces BDO, also provided herein is a NNOMO comprising at least oneexogenous nucleic acid encoding a BDOPE, where the microbial organismproduces a BDOP intermediate, such as succinyl-CoA, succinatesemialdehyde, 4-HB, 4-hydroxybutyryl-phosphate, 4-hydroxybutyryl-CoA or4-hydroxybutanal.

In some embodiments, the carbon feedstock and other cellular uptakesources such as phosphate, ammonia, sulfate, chloride and other halogenscan be chosen to alter the isotopic distribution of the atoms present inBDO and/or 4-HB or any BDO and/or 4-HB pathway intermediate. The variouscarbon feedstock and other uptake sources enumerated above will bereferred to herein, collectively, as “uptake sources.” Uptake sourcescan provide isotopic enrichment for any atom present in the product BDOand/or 4-HB or BDO and/or 4-HB pathway intermediate, or for sideproducts generated in reactions diverging away from a BDO and/or 4-HBpathway. Isotopic enrichment can be achieved for any target atomincluding, for example, carbon, hydrogen, oxygen, nitrogen, sulfur,phosphorus, chloride or other halogens. The same holds true for the MMPsand FAPs, as well as intermediates thereof, provided herein.

In some embodiments, the uptake sources can be selected to alter thecarbon-12, carbon-13, and carbon-14 ratios. In some embodiments, theuptake sources can be selected to alter the oxygen-16, oxygen-17, andoxygen-18 ratios. In some embodiments, the uptake sources can beselected to alter the hydrogen, deuterium, and tritium ratios. In someembodiments, the uptake sources can selected to alter the nitrogen-14and nitrogen-15 ratios. In some embodiments, the uptake sources can beselected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35ratios. In some embodiments, the uptake sources can be selected to alterthe phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In someembodiments, the uptake sources can be selected to alter thechlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be variedto a desired ratio by selecting one or more uptake sources. An uptakesource can be derived from a natural source, as found in nature, or froma man-made source, and one skilled in the art can select a naturalsource, a man-made source, or a combination thereof, to achieve adesired isotopic ratio of a target atom. An example of a man-made uptakesource includes, for example, an uptake source that is at leastpartially derived from a chemical synthetic reaction. Such isotopicallyenriched uptake sources can be purchased commercially or prepared in thelaboratory and/or optionally mixed with a natural source of the uptakesource to achieve a desired isotopic ratio. In some embodiments, atarget isotopic ratio of an uptake source can be obtained by selecting adesired origin of the uptake source as found in nature. For example, asdiscussed herein, a natural source can be a biobased derived from orsynthesized by a biological organism or a source such as petroleum-basedproducts or the atmosphere. In some such embodiments, a source ofcarbon, for example, can be selected from a fossil fuel-derived carbonsource, which can be relatively depleted of carbon-14, or anenvironmental or atmospheric carbon source, such as CO₂, which canpossess a larger amount of carbon-14 than its petroleum-derivedcounterpart.

Isotopic enrichment is readily assessed by mass spectrometry usingtechniques known in the art such as Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC) and/or high performance liquid chromatography (HPLC).

The unstable carbon isotope carbon-14 or radiocarbon makes up forroughly 1 in 10¹² carbon atoms in the earth's atmosphere and has ahalf-life of about 5700 years. The stock of carbon is replenished in theupper atmosphere by a nuclear reaction involving cosmic rays andordinary nitrogen (¹⁴N). Fossil fuels contain no carbon-14, as itdecayed long ago. Burning of fossil fuels lowers the atmosphericcarbon-14 fraction, the so-called “Suess effect”.

Methods of determining the isotopic ratios of atoms in a compound arewell known to those skilled in the art. Isotopic enrichment is readilyassessed by mass spectrometry using techniques known in the art such asaccelerated mass spectrometry (AMS), Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC), high performance liquid chromatography (HPLC) and/or gaschromatography, and the like.

In the case of carbon, ASTM D6866 was developed in the United States asa standardized analytical method for determining the biobased content ofsolid, liquid, and gaseous samples using radiocarbon dating by theAmerican Society for Testing and Materials (ASTM) International. Thestandard is based on the use of radiocarbon dating for the determinationof a product's biobased content. ASTM D6866 was first published in 2004,and the current active version of the standard is ASTM D6866-11(effective Apr. 1, 2011). Radiocarbon dating techniques are well knownto those skilled in the art, including those described herein.

The biobased content of a compound is estimated by the ratio ofcarbon-14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modern(Fm) is computed from the expression: Fm=(S−B)/(M−B), where B, S and Mrepresent the ¹⁴C/¹²C ratios of the blank, the sample and the modernreference, respectively. Fraction Modern is a measurement of thedeviation of the ¹⁴C/¹²C ratio of a sample from “Modern.” Modern isdefined as 95% of the radiocarbon concentration (in AD 1950) of NationalBureau of Standards (NBS) Oxalic Acid I (i.e., standard referencematerials (SRM) 4990b) normalized to δ¹³C_(VPDB)=−19 per mil (Olsson,The use of Oxalic acid as a Standard. in, Radiocarbon Variations andAbsolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, NewYork (1970)). Mass spectrometry results, for example, measured by ASM,are calculated using the internationally agreed upon definition of 0.95times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalizedto δ¹³C_(VPDB)=−19 per mil. This is equivalent to an absolute (AD1950)¹⁴C/¹²C ratio of 1.176±0.010×10¹² (Karlen et al., Arkiv Geoftsik,4:465-471 (1968)). The standard calculations take into account thedifferential uptake of one isotope with respect to another, for example,the preferential uptake in biological systems of C¹² over C¹³ over C¹⁴,and these corrections are reflected as a Fm corrected for δ¹³.

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of1955 sugar beet. Although there were 1000 lbs made, this oxalic acidstandard is no longer commercially available. The Oxalic Acid IIstandard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of1977 French beet molasses. In the early 1980's, a group of 12laboratories measured the ratios of the two standards. The ratio of theactivity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). Theisotopic ratio of HOx II is −17.8 per mille. ASTM D6866-11 suggests useof the available Oxalic Acid II standard SRM 4990 C (Hox2) for themodern standard (see discussion of original vs. currently availableoxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). AFm=0% represents the entire lack of carbon-14 atoms in a material, thusindicating a fossil (for example, petroleum based) carbon source. AFm=100%, after correction for the post-1950 injection of carbon-14 intothe atmosphere from nuclear bomb testing, indicates an entirely moderncarbon source. As described herein, such a “modern” source includesbiobased sources.

As described in ASTM D6866, the percent modern carbon (pMC) can begreater than 100% because of the continuing but diminishing effects ofthe 1950s nuclear testing programs, which resulted in a considerableenrichment of carbon-14 in the atmosphere as described in ASTM D6866-11.Because all sample carbon-14 activities are referenced to a “pre-bomb”standard, and because nearly all new biobased products are produced in apost-bomb environment, all pMC values (after correction for isotopicfraction) must be multiplied by 0.95 (as of 2010) to better reflect thetrue biobased content of the sample. A biobased content that is greaterthan 103% suggests that either an analytical error has occurred, or thatthe source of biobased carbon is more than several years old.

ASTM D6866 quantifies the biobased content relative to the material'stotal organic content and does not consider the inorganic carbon andother non-carbon containing substances present. For example, a productthat is 50% starch-based material and 50% water would be considered tohave a Biobased Content=100% (50% organic content that is 100% biobased)based on ASTM D6866. In another example, a product that is 50%starch-based material, 25% petroleum-based, and 25% water would have aBiobased Content=66.7% (75% organic content but only 50% of the productis biobased). In another example, a product that is 50% organic carbonand is a petroleum-based product would be considered to have a BiobasedContent=0% (50% organic carbon but from fossil sources). Thus, based onthe well known methods and known standards for determining the biobasedcontent of a compound or material, one skilled in the art can readilydetermine the biobased content and/or prepared downstream productshaving a desired biobased content.

Applications of carbon-14 dating techniques to quantify bio-basedcontent of materials are known in the art (Currie et al., NuclearInstruments and Methods in Physics Research B, 172:281-287 (2000)). Forexample, carbon-14 dating has been used to quantify bio-based content interephthalate-containing materials (Colonna et al., Green Chemistry,13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT)polymers derived from renewable 1,3-propanediol and petroleum-derivedterephthalic acid resulted in Fm values near 30% (i.e., since 3/11 ofthe polymeric carbon derives from renewable 1,3-propanediol and 8/11from the fossil end member terephthalic acid) (Currie et al., supra,2000). In contrast, polybutylene terephthalate polymer derived from bothrenewable BDO and renewable terephthalic acid resulted in bio-basedcontent exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, provided are BDO and/or 4-HB or a BDOand/or 4-HB pathway intermediate thereof that has a carbon-12,carbon-13, and carbon-14 ratio that reflects an atmospheric carbon, alsoreferred to as environmental carbon, uptake source. For example, in someaspects the BDO and/or 4-HB or a BDO and/or 4-HB intermediate thereofcan have an Fm value of at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98% or as much as 100%. In some such embodiments, the uptakesource is CO₂. In some embodiments, provided is BDO and/or 4-HB or a BDOand/or 4-HB intermediate thereof that has a carbon-12, carbon-13, andcarbon-14 ratio that reflects petroleum-based carbon uptake source. Inthis aspect, the BDO and/or 4-HB or a BDO and/or 4-HB intermediatethereof can have an Fm value of less than 95%, less than 90%, less than85%, less than 80%, less than 75%, less than 70%, less than 65%, lessthan 60%, less than 55%, less than 50%, less than 45%, less than 40%,less than 35%, less than 30%, less than 25%, less than 20%, less than15%, less than 10%, less than 5%, less than 2% or less than 1%. In someembodiments, provided is BDO and/or 4-HB or a BDO and/or 4-HBintermediate thereof that has a carbon-12, carbon-13, and carbon-14ratio that is obtained by a combination of an atmospheric carbon uptakesource with a petroleum-based uptake source. Using such a combination ofuptake sources is one way by which the carbon-12, carbon-13, andcarbon-14 ratio can be varied, and the respective ratios would reflectthe proportions of the uptake sources.

Further, the present invention relates, in part, to biologicallyproduced BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof asdisclosed herein, and to the products derived therefrom, wherein the BDOand/or 4-HB or a BDO and/or 4-HB intermediate thereof has a carbon-12,carbon-13, and carbon-14 isotope ratio of about the same value as theCO₂ that occurs in the environment. For example, in some aspects,provided are a bioderived BDO and/or 4-HB or a bioderived BDO and/or4-HB intermediate thereof having a carbon-12 versus carbon-13 versuscarbon-14 isotope ratio of about the same value as the CO₂ that occursin the environment, or any of the other ratios disclosed herein. It isunderstood, as disclosed herein, that a product can have a carbon-12versus carbon-13 versus carbon-14 isotope ratio of about the same valueas the CO₂ that occurs in the environment, or any of the ratiosdisclosed herein, wherein the product is generated from bioderived BDOand/or 4-HB or a bioderived BDO and/or 4-HB intermediate thereof asdisclosed herein, wherein the bioderived product is chemically modifiedto generate a final product. Methods of chemically modifying abioderived product of BDO and/or 4-HB, or an intermediate thereof, togenerate a desired product are well known to those skilled in the art,as described herein. Also provided are plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such aspoly-4-hydroxybutyrate (P4HB) or co-polymers thereof,poly(tetramethylene ether) glycol (PTMEG)(also referred to as PTMO,polytetramethylene oxide) and polyurethane-polyurea copolymers, referredto as spandex, elastane or Lycra™, nylons, and the like, having acarbon-12 versus carbon-13 versus carbon-14 isotope ratio of about thesame value as the CO₂ that occurs in the environment, wherein theplastics, elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG andpolyurethane-polyurea copolymers, referred to as spandex, elastane orLycra™, nylons, and the like, are generated directly from or incombination with bioderived BDO and/or 4-HB or a bioderived BDO and/or4-HB intermediate thereof as disclosed herein.

BDO and/or 4-HB are chemicals used in commercial and industrialapplications. Non-limiting examples of such applications includeproduction of plastics, elastic fibers, polyurethanes, polyesters,including polyhydroxyalkanoates such as P4HB or co-polymers thereof,PTMEG and polyurethane-polyurea copolymers, referred to as spandex,elastane or Lycra™, nylons, and the like. Moreover, BDO and/or 4-HB arealso used as a raw material in the production of a wide range ofproducts including plastics, elastic fibers, polyurethanes, polyesters,including polyhydroxyalkanoates such as P4HB or co-polymers thereof,PTMEG and polyurethane-polyurea copolymers, referred to as spandex,elastane or Lycra™, nylons, and the like. Accordingly, in someembodiments, provided are biobased plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HBor co-polymers thereof, PTMEG and polyurethane-polyurea copolymers,referred to as spandex, elastane or Lycra™, nylons, and the like,comprising one or more bioderived BDO and/or 4-HB or bioderived BDOand/or 4-HB intermediate thereof produced by a NNOMO provided herein orproduced using a method disclosed herein.

As used herein, the term “bioderived” means derived from or synthesizedby a biological organism and can be considered a renewable resourcesince it can be generated by a biological organism. Such a biologicalorganism, in particular the microbial organisms of the inventiondisclosed herein, can utilize feedstock or biomass, such as, sugars orcarbohydrates obtained from an agricultural, plant, bacterial, or animalsource. Alternatively, the biological organism can utilize atmosphericcarbon. As used herein, the term “biobased” means a product as describedabove that is composed, in whole or in part, of a bioderived compound ofthe invention. A biobased or bioderived product is in contrast to apetroleum derived product, wherein such a product is derived from orsynthesized from petroleum or a petrochemical feedstock.

In some embodiments, the invention provides plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HBor co-polymers thereof, PTMEG and polyurethane-polyurea copolymers,referred to as spandex, elastane or Lycra™, nylons, and the like,comprising bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HBintermediate thereof, wherein the bioderived BDO and/or 4-HB orbioderived BDO and/or 4-HB intermediate thereof includes all or part ofthe BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof used in theproduction of plastics, elastic fibers, polyurethanes, polyesters,including polyhydroxyalkanoates such as P4HB or co-polymers thereof,PTMEG and polyurethane-polyurea copolymers, referred to as spandex,elastane or Lycra″, nylons, and the like. Thus, in some aspects, theinvention provides a biobased plastics, elastic fibers, polyurethanes,polyesters, including polyhydroxyalkanoates such as P4HB or co-polymersthereof, PTMEG and polyurethane-polyurea copolymers, referred to asspandex, elastane or Lycra™, nylons, and the like, comprising at least2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98% or 100% bioderived BDO and/or 4-HB or bioderived BDO and/or4-HB intermediate thereof as disclosed herein. Additionally, in someaspects, the invention provides a biobased plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HBor co-polymers thereof, PTMEG and polyurethane-polyurea copolymers,referred to as spandex, elastane or Lycra″, nylons, and the like,wherein the BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof usedin its production is a combination of bioderived and petroleum derivedBDO and/or 4-HB or BDO and/or 4-HB intermediate thereof. For example, abiobased plastics, elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG andpolyurethane-polyurea copolymers, referred to as spandex, elastane orLycra™, nylons, and the like, can be produced using 50% bioderived BDOand/or 4-HB and 50% petroleum derived BDO and/or 4-HB or other desiredratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%,40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derivedprecursors, so long as at least a portion of the product comprises abioderived product produced by the microbial organisms disclosed herein.It is understood that methods for producing plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HBor co-polymers thereof, PTMEG and polyurethane-polyurea copolymers,referred to as spandex, elastane or Lycra™, nylons, and the like, usingthe bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HBintermediate thereof of the invention are well known in the art.

In one embodiment, the product is a plastic. In one embodiment, theproduct is an elastic fiber. In one embodiment, the product is apolyurethane. In one embodiment, the product is a polyester. In oneembodiment, the product is a polyhydroxyalkanoate. In one embodiment,the product is a poly-4-HB. In one embodiment, the product is aco-polymer of poly-4-HB. In one embodiment, the product is apoly(tetramethylene ether) glycol. In one embodiment, the product is apolyurethane-polyurea copolymer. In one embodiment, the product is aspandex. In one embodiment, the product is an elastane. In oneembodiment, the product is a Lycra™. In one embodiment, the product is anylon.

In some embodiments, provided herein is a culture medium comprisingbioderived BDO. In some embodiments, the bioderived BDO is produced byculturing a NNOMO having a MMP and BDOP, as provided herein. In certainembodiments, the bioderived BDO has a carbon-12, carbon-13 and carbon-14isotope ratio that reflects an atmospheric carbon dioxide uptake source.In one embodiment, the culture medium is separated from a NNOMO having aMMP and BDOP.

In other embodiments, provided herein is a bioderived BDO. In someembodiments, the bioderived BDO is produced by culturing a NNOMO havinga MMP and BDOP, as provided herein. In certain embodiments, thebioderived BDO has a carbon-12, carbon-13 and carbon-14 isotope ratiothat reflects an atmospheric carbon dioxide uptake source. In someembodiments, the bioderived BDO has an Fm value of at least 80%, atleast 85%, at least 90%, at least 95% or at least 98%. In certainembodiments, the bioderived BDO is a component of culture medium.

In certain embodiments, provided herein is a composition comprising abioderived BDO provided herein, for example, a bioderived BDO producedby culturing a NNOMO having a MMP and BDOP, as provided herein. In someembodiments, the composition further comprises a compound other thansaid bioderived BDO. In certain embodiments, the compound other thansaid bioderived BDO is a trace amount of a cellular portion of a NNOMOhaving a MMP and a BDOP, as provided herein.

In some embodiments, provided herein is a biobased product comprising abioderived BDO provided herein. In certain embodiments, the biobasedproduct is a plastic, elastic fiber, polyurethane, polyester,polyhydroxyalkanoate, poly-4-HB, co-polymer of poly-4-HB,poly(tetramethylene ether) glycol, polyurethane-polyurea copolymer,spandex, elastane, Lycra™, or nylon. In certain embodiments, thebiobased product comprises at least 5% bioderived BDO. In certainembodiments, the biobased product is (i) a polymer, THF or a THFderivative, or GBL or a GBL derivative; (ii) a plastic, elastic fiber,polyurethane, polyester, polyhydroxyalkanoate, poly-4-HB, co-polymer ofpoly-4-HB, poly(tetramethylene ether) glycol, polyurethane-polyureacopolymer, spandex, elastane, Lycra™, or nylon; (iii) a polymer, aresin, a fiber, a bead, a granule, a pellet, a chip, a plastic, apolyester, a thermoplastic polyester, a molded article, aninjection-molded article, an injection-molded part, an automotive part,an extrusion resin, an electrical part and a casing; and optionallywhere the biobased product is reinforced or filled and further where thebiobased product is glass-reinforced or -filled or mineral-reinforced or-filled; (iv) a polymer, wherein the polymer comprises polybutyleneterephthalate (PBT); (v) a polymer, wherein the polymer comprises PBTand the biobased product is a resin, a fiber, a bead, a granule, apellet, a chip, a plastic, a polyester, a thermoplastic polyester, amolded article, an injection-molded article, an injection-molded part,an automotive part, an extrusion resin, an electrical part and a casing;and optionally where the biobased product is reinforced or filled andfurther where the biobased product is glass-reinforced or—filled ormineral-reinforced or -filled; (vi) a THF or a THF derivative, whereinthe THF derivative is polytetramethylene ether glycol (PTMEG), apolyester ether (COPE) or a thermoplastic polyurethane; (viii) a THFderivative, wherein the THF derivative comprises a fiber; or (ix) a GBLor a GBL derivative, wherein the GBL derivative is a pyrrolidone. Incertain embodiments, the biobased product comprises at least 10%bioderived BDO. In some embodiments, the biobased product comprises atleast 20% bioderived BDO. In other embodiments, the biobased productcomprises at least 30% bioderived BDO. In some embodiments, the biobasedproduct comprises at least 40% bioderived BDO. In other embodiments, thebiobased product comprises at least 50% bioderived BDO. In oneembodiment, the biobased product comprises a portion of said bioderivedBDO as a repeating unit. In another embodiment, provided herein is amolded product obtained by molding the biobased product provided herein.In other embodiments, provided herein is a process for producing abiobased product provided herein, comprising chemically reacting saidbioderived-BDO with itself or another compound in a reaction thatproduces said biobased product. In certain embodiments, provided hereinis a polymer comprising or obtained by converting the bioderived BDO. Inother embodiments, provided herein is a method for producing a polymer,comprising chemically or enzymatically converting the bioderived BDO tothe polymer. In yet other embodiments, provided herein is a compositioncomprising the bioderived BDO, or a cell lysate or culture supernatantthereof.

In some embodiments, provided herein is a culture medium comprisingbioderived 4-HB. In some embodiments, the bioderived 4-HB is produced byculturing a NNOMO having a MMP and BDO and/or 4-HB pathway, as providedherein. In certain embodiments, the bioderived 4-HB has a carbon-12,carbon-13 and carbon-14 isotope ratio that reflects an atmosphericcarbon dioxide uptake source. In one embodiment, the culture medium isseparated from a NNOMO having a MMP and BDO and/or 4-HB pathway.

In other embodiments, provided herein is a bioderived 4-HB. In someembodiments, the bioderived 4-HB is produced by culturing a NNOMO havinga MMP and BDO and/or 4-HB pathway, as provided herein. In certainembodiments, the bioderived 4-HB has a carbon-12, carbon-13 andcarbon-14 isotope ratio that reflects an atmospheric carbon dioxideuptake source. In some embodiments, the bioderived 4-HB has an Fm valueof at least 80%, at least 85%, at least 90%, at least 95% or at least98%. In certain embodiments, the bioderived 4-HB is a component ofculture medium.

In certain embodiments, provided herein is a composition comprising abioderived 4-HB provided herein, for example, a bioderived 4-HB producedby culturing a NNOMO having a MMP and BDO and/or 4-HB pathway, asprovided herein. In some embodiments, the composition further comprisesa compound other than said bioderived 4-HB. In certain embodiments, thecompound other than said bioderived 4-HB is a trace amount of a cellularportion of a NNOMO having a MMP and a BDO and/or 4-HB pathway, asprovided herein.

In some embodiments, provided herein is a biobased product comprising abioderived 4-HB provided herein. In certain embodiments, the biobasedproduct is a plastic, elastic fiber, polyurethane, polyester,polyhydroxyalkanoate, poly-4-HB, co-polymer of poly-4-HB,poly(tetramethylene ether) glycol, polyurethane-polyurea copolymer,spandex, elastane, Lycra™, or nylon. In certain embodiments, thebiobased product comprises at least 5% bioderived 4-HB. In certainembodiments, the biobased product comprises at least 10% bioderived4-HB. In some embodiments, the biobased product comprises at least 20%bioderived 4-HB. In other embodiments, the biobased product comprises atleast 30% bioderived 4-HB. In some embodiments, the biobased productcomprises at least 40% bioderived 4-HB. In other embodiments, thebiobased product comprises at least 50% bioderived 4-HB. In oneembodiment, the biobased product comprises a portion of said bioderived4-HB as a repeating unit. In another embodiment, provided herein is amolded product obtained by molding the biobased product provided herein.In other embodiments, provided herein is a process for producing abiobased product provided herein, comprising chemically reacting saidbioderived 4-HB with itself or another compound in a reaction thatproduces said biobased product.

Also provided herein is a method of producing formaldehyde, comprisingculturing a NNOMO provided herein (e.g., comprising an exogenous nucleicacid encoding an EM9 (1J)) under conditions and for a sufficient periodof time to produce formaldehyde. In certain embodiments, theformaldehyde is consumed to provide a reducing equivalent. In otherembodiments, the formaldehyde is consumed to incorporate into BDO. Inyet other embodiments, the formaldehyde is consumed to incorporate intoanother target product.

Also provided herein is a method of producing an intermediate ofglycolysis and/or an intermediate of a metabolic pathway that can beused in the formation of biomass, comprising culturing a NNOMO providedherein (e.g., comprising an exogenous nucleic acid encoding an EM9 (1J))under conditions and for a sufficient period of time to produce theintermediate. In one embodiment, the method is a method of producing anintermediate of glycolysis. In other embodiments, the method is a methodof producing an intermediate of a metabolic pathway that can be used inthe formation of biomass. In certain embodiments, the intermediate isconsumed to provide a reducing equivalent. In other embodiment, theintermediate is consumed to incorporate into BDO. In yet otherembodiments, the formaldehyde is consumed to incorporate into anothertarget product.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reactionand that reference to any of these metabolic constituents alsoreferences the gene or genes encoding the enzymes that catalyze, orproteins involved in, the referenced reaction, reactant or product.Likewise, given the well known fields of metabolic biochemistry,enzymology and genomics, reference herein to a gene or encoding nucleicacid also constitutes a reference to the corresponding encoded enzymeand the reaction it catalyzes, or a protein associated with thereaction, as well as the reactants and products of the reaction.

The production of 4-HB via biosynthetic modes using the microbialorganisms of the invention is particularly useful because it can producemonomeric 4-HB. The NNOMOs of the invention and their biosynthesis of4-HB and BDO family compounds also is particularly useful because the4-HB product can be (1) secreted; (2) can be devoid of anyderivatizations such as Coenzyme A; (3) avoids thermodynamic changesduring biosynthesis; (4) allows direct biosynthesis of BDO, and (5)allows for the spontaneous chemical conversion of 4-HB toγ-butyrolactone (GBL) in acidic pH medium. This latter characteristicalso is particularly useful for efficient chemical synthesis orbiosynthesis of BDO family compounds such as BDO and/or tetrahydrofuran(THF), for example.

Microbial organisms generally lack the capacity to synthesize 4-HB andtherefore any of the compounds disclosed herein to be within the BDOfamily of compounds or known by those in the art to be within the BDOfamily of compounds. Moreover, organisms having all of the requisitemetabolic enzymatic capabilities are not known to produce 4-HB from theenzymes described and biochemical pathways exemplified herein. Rather,with the possible exception of a few anaerobic microorganisms describedfurther below, the microorganisms having the enzymatic capability to use4-HB as a substrate to produce, for example, succinate. In contrast, theNNOMOs of the invention can generate BDO and/or 4-HB as a product. Thebiosynthesis of 4-HB in its monomeric form is not only particularlyuseful in chemical synthesis of BDO family of compounds, it also allowsfor the further biosynthesis of BDO family compounds and avoidsaltogether chemical synthesis procedures.

The NNOMOs of the invention that can produce BDO and/or 4-HB areproduced by ensuring that a host microbial organism includes functionalcapabilities for the complete biochemical synthesis of at least one BDOand/or 4-HB biosynthetic pathway of provided herein. Ensuring at leastone requisite BDO and/or 4-HB biosynthetic pathway confers BDO and/or4-HB biosynthesis capability onto the host microbial organism.

The organisms and methods are described herein with general reference tothe metabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

The NNOMOs described herein can be produced by introducing expressiblenucleic acids encoding one or more of the enzymes or proteinsparticipating in one or more methanol metabolic, formaldehydeassimilation and/or BDO biosynthetic pathways. Depending on the hostmicrobial organism chosen for biosynthesis, nucleic acids for some orall of a particular methanol metabolic, formaldehyde assimilation and/orBDO biosynthetic pathway can be expressed. For example, if a chosen hostis deficient in one or more enzymes or proteins for a desired metabolic,assimilation or biosynthetic pathway, then expressible nucleic acids forthe deficient enzyme(s) or protein(s) are introduced into the host forsubsequent exogenous expression. Alternatively, if the chosen hostexhibits endogenous expression of some pathway genes, but is deficientin others, then an encoding nucleic acid is needed for the deficientenzyme(s) or protein(s) to achieve BDO biosynthesis and/or methanolmetabolism. Thus, a NNOMO described herein can be produced byintroducing exogenous enzyme or protein activities to obtain a desiredmetabolic pathway or biosynthetic pathway, and/or a desired metabolicpathway or biosynthetic pathway can be obtained by introducing one ormore exogenous enzyme or protein activities that, together with one ormore endogenous enzymes or proteins, produces a desired product such asBDO.

Host microbial organisms can be selected from, and the NNOMOs generatedin, for example, bacteria, yeast, fungus or any of a variety of othermicroorganisms applicable or suitable to fermentation processes.Exemplary bacteria include any species selected from the orderEnterobacteriales, family Enterobacteriaceae, including the generaEscherichia and Klebsiella; the order Aeromonadales, familySuccinivibrionaceae, including the genus Anaerobiospirillum; the orderPasteurellales, family Pasteurellaceae, including the generaActinobacillus and Mannheimia; the order Rhizobiales, familyBradyrhizobiaceae, including the genus Rhizobium; the order Bacillales,family Bacillaceae, including the genus Bacillus; the orderActinomycetales, families Corynebacteriaceae and Streptomycetaceae,including the genus Corynebacterium and the genus Streptomyces,respectively; order Rhodospirillales, family Acetobacteraceae, includingthe genus Gluconobacter; the order Sphingomonadales, familySphingomonadaceae, including the genus Zymomonas; the orderLactobacillales, families Lactobacillaceae and Streptococcaceae,including the genus Lactobacillus and the genus Lactococcus,respectively; the order Clostridiales, family Clostridiaceae, genusClostridium; and the order Pseudomonadales, family Pseudomonadaceae,including the genus Pseudomonas. Non-limiting species of host bacteriainclude Escherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.

Similarly, exemplary species of yeast or fungi species include anyspecies selected from the order Saccharomycetales, familySaccaromycetaceae, including the genera Saccharomyces, Kluyveromyces andPichia; the order Saccharomycetales, family Dipodascaceae, including thegenus Yarrowia; the order Schizosaccharomycetales, familySchizosaccaromycetaceae, including the genus Schizosaccharomyces; theorder Eurotiales, family Trichocomaceae, including the genusAspergillus; and the order Mucorales, family Mucoraceae, including thegenus Rhizopus. Non-limiting species of host yeast or fungi includeSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger,Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowialipolytica, and the like. E. coli is a particularly useful host organismsince it is a well characterized microbial organism suitable for geneticengineering. Other particularly useful host organisms include yeast suchas Saccharomyces cerevisiae. It is understood that any suitablemicrobial host organism can be used to introduce metabolic and/orgenetic modifications to produce a desired product.

In some embodiments, the host microbial organism can be a recombinantmicrobial organism having increased succinate (succinic acid) productionas compared to the wild-type microbial organism. Increased succinateproduction can be generated by introduction of one or more genedisruptions of a host microbial organism gene and/or an exogenousnucleic acid. Methods of increasing succinate production in a microbialorganism are well known in the art. For example, the host microbialorganism can be a recombinant bacteria, such as a rumen bacteria, thatincludes a gene disruption in one or more genes selected from a lactatedehydrogenase gene (ldhA), a pyruvate formate-lyase gene (pfl), aphosphotransacetylase gene (pta), and an acetate kinase gene (ackA) asdescribed in U.S. Publication 2007-0054387, published Mar. 8, 2007, nowU.S. Pat. No. 7,470,530, and U.S. Publication 2009-0203095, publishedAug. 13, 2009. For example, in one aspect, the host microbial organismcan include a gene disruption in a gene encoding ldhA, pta, and ackA,without disrupting a gene encoding pfl. Accordingly, in some aspects,the bacteria that can be used as a host microbial organism include, butare not limited to, a Mannheimia species (e.g., Mannheimia sp. LPK,Mannheimia sp. LPK4, Mannheimia sp. LPK7, Mannheimia sp. LPK (KCTC10558BP), Mannheimia succiniciproducens MBEL55E (KCTC 0769BP),Mannheimia succiniciproducens PALK (KCTC10973BP), Mannheimiasucciniciproducens ALK, or Mannheimia succiniciproducens ALKt), anActinobacillus species (e.g., Actinobacillus succinogenes), aBacteroides species, a Succinimonas species, a Succinivibrio species, oran Anaerobiospirillum species (e.g., Anaerobiospirillumsucciniciproducens).

Additional methods for producing a host microbial organism havingincreased succinate production are also well known in the art. Forexample, the host microbial organism can have genes disruptions in genesencoding ldhA, pfl and a phosphopyruvate carboxylase (ppc), oralternatively/additionally gene disruptions in genes encoding a glucosephosphotransferase (ptsG) and a pyruvate kinase (pykA and pykF), oralternatively/additionally gene disruptions in a gene encoding asuccinic semialdehyde dehydrogenase (GabD), oralternatively/additionally introduction or amplification of a nucleicacid encoding a C4-dicarboxylate transport protein (DctA), which isassociated with transport of succinate, as described in U.S. Publication2010-0330634, published Dec. 30, 2010. Accordingly, a host microbialorganism can include a Lumen bacteria, a Corynebacterium species, aBrevibacterium species or an Escherichia species (e.g., Escherichiacoli, in particular strain W3110GFA, as disclosed in U.S. Publication2009-0075352, published Mar. 19, 2009). As yet another example, a hostmicrobial organism having increased succinate production can begenerated by introducing an exogenous nucleic acid encoding an enzyme orprotein that increases production of succinate are described in U.S.Publication 2007-0042476, published Feb. 22, 2007, U.S. Publication2007-0042477, published Feb. 22, 2007, and U.S. Publication2008-0020436, published Jan. 24, 2008, which disclose introduction of anucleic acid encoding a malic enzyme B (maeB), a fumarate hydratase C(fumC), a formate dehydrogenase D (fdhD) or a formate dehydrogenase E(fdhE). Additional useful host microbial organisms include, but are notlimited to, a microbial organism that can produce succinate usingglycerol as a carbon source, as disclosed in WO 2009/048202, or anorganism that simultaneously use sucrose and glycerol as carbon sourcesto produce succinate by weakening a catabolic inhibition mechanism ofthe glycerol by sucrose as described in EP 2612905.

Additional microbes having high succinate production suitable for use asa host microbial organism for the pathways and methods described hereininclude those bacterial strains described in International PublicationsWO 2010/092155 and WO 2009/024294, and U.S. Publication 2010-0159542,published Jun. 24, 2010. For example, bacterial strains of the genusPasteurella, which are gram negative, facultative anaerobes, motile,pleimorphic and often catalase- and oxidase-positive, specificallyPasteurella strain DD1 and its variants, are suitable host microbialorganisms. Pasteurella strain DD1 is the bacterial strain depositedunder the Budapest Treaty with DSMZ (Deutsche SammlungvonMikroorganismen and Zellkulturen, GmbH), Germany, having deposit numberDSM18541, and was originally isolated from the rumen of a cow of Germanorigin. Improved variants of DD1, are described in WO 2010/092155, arealso suitable host microbial organisms, and include, but art not limitedto, LU15348 (DD1 with deletion of pfl gene); LU15050 (DD1 deletion ofldh gene); and LU15224 (DD1 with deletion of both pfl and ldh genes).Additional host bacteria include succinate-producers isolated frombovine rumen belonging to the genus Mannheimia, specifically the speciesMannheimia succiniciproducens, and strain Mannheimia succiniciproducensMBEL55E and its variants.

Depending on the BDO biosynthetic, methanol metabolic and/or FAPconstituents of a selected host microbial organism, the NNOMOs providedherein will include at least one exogenously expressed BDO, formaldehydeassimilation and/or MMP-encoding nucleic acid and up to all encodingnucleic acids for one or more BDO biosynthetic pathways, FAPs and/orMMPs. For example, BDO biosynthesis can be established in a hostdeficient in a pathway enzyme or protein through exogenous expression ofthe corresponding encoding nucleic acid. In a host deficient in allenzymes or proteins of a BDOP, exogenous expression of all enzyme orproteins in the pathway can be included, although it is understood thatall enzymes or proteins of a pathway can be expressed even if the hostcontains at least one of the pathway enzymes or proteins. For example,exogenous expression of all enzymes or proteins in a pathway forproduction of BDO can be included. The same holds true for the MMPs andFAPs provided herein.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the BDOP, FAP,and MMP deficiencies of the selected host microbial organism. Therefore,a NNOMO of the invention can have one, two, three, four, five, six,seven, eight, or up to all nucleic acids encoding the enzymes orproteins constituting a MMP, formaldehyde assimilation and/or BDObiosynthetic pathway disclosed herein. In some embodiments, the NNOMOsalso can include other genetic modifications that facilitate or optimizeBDO biosynthesis, formaldehyde assimilation and/or methanol metabolismor that confer other useful functions onto the host microbial organism.One such other functionality can include, for example, augmentation ofthe synthesis of one or more of the BDOP precursors, such asalpha-ketoglutarate, succinate, fumarate, oxaloacetate,phosphoenolpyruvate, or any combination thereof.

Generally, a host microbial organism is selected such that it producesthe precursor of a BDOP, either as a naturally produced molecule or asan engineered product that either provides de novo production of adesired precursor or increased production of a precursor naturallyproduced by the host microbial organism. A host organism can beengineered to increase production of a precursor, as disclosed herein.In addition, a microbial organism that has been engineered to produce adesired precursor can be used as a host organism and further engineeredto express enzymes or proteins of a BDOP.

In some embodiments, a NNOMO provided herein is generated from a hostthat contains the enzymatic capability to synthesize BDO, assimilateformaldehyde and/or metabolize methanol. In this specific embodiment itcan be useful to increase the synthesis or accumulation of a BDOPproduct, FAP product and/or MMP product (e.g., reducing equivalentsand/or formaldehyde) to, for example, drive BDOP reactions toward BDOproduction. Increased synthesis or accumulation can be accomplished by,for example, overexpression of nucleic acids encoding one or more of theabove-described BDO, formaldehyde assimilation and/or MMP enzymes orproteins. Over expression the enzyme(s) and/or protein(s) of the BDOP,formaldehyde assimilation, and/or MMP can occur, for example, throughexogenous expression of the endogenous gene(s), or through exogenousexpression of the heterologous gene(s). Therefore, naturally occurringorganisms can be readily generated to be NNOMOs, for example, producingBDO through overexpression of one, two, three, four, five, six, seven,eight, up to all nucleic acids encoding BDO biosynthetic pathway, and/orMMP enzymes or proteins. Naturally occurring organisms can also bereadily generated to be NNOMOs, for example, assimilating formaldehyde,through overexpression of one, two, three, four, five, six, seven,eight, up to all nucleic acids encoding FAP, and/or MMP enzymes orproteins. In addition, a N can be generated by mutagenesis of anendogenous gene that results in an increase in activity of an enzyme inthe BDO biosynthetic, formaldehyde assimilation and/or methanolmetabolic pathway(s).

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into a NNOMO.

It is understood that, in methods provided herein, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a NNOMO provided herein. The nucleic acids can be introducedso as to confer, for example, a BDO biosynthetic, formaldehydeassimilation and/or methanol metabolic pathway onto the microbialorganism. Alternatively, encoding nucleic acids can be introduced toproduce an intermediate microbial organism having the biosyntheticcapability to catalyze some of the required reactions to confer BDObiosynthetic, formaldehyde assimilation and/or methanol metaboliccapability. For example, a NNOMO having a BDOP, FAP and/or MMP cancomprise at least two exogenous nucleic acids encoding desired enzymesor proteins. Thus, it is understood that any combination of two or moreenzymes or proteins of a biosynthetic pathway, FAP and/or metabolicpathway can be included in a NNOMO provided herein. Similarly, it isunderstood that any combination of three or more enzymes or proteins ofa biosynthetic pathway, FAP and/or metabolic pathway can be included ina NNOMO provided herein, as desired, so long as the combination ofenzymes and/or proteins of the desired biosynthetic pathway, FAP and/ormetabolic pathway results in production of the corresponding desiredproduct. Similarly, any combination of four or more enzymes or proteinsof a biosynthetic pathway, FAP and/or MMP as disclosed herein can beincluded in a NNOMO provided herein, as desired, so long as thecombination of enzymes and/or proteins of the desired biosynthetic,assimilation and/or metabolic pathway results in production of thecorresponding desired product. In specific embodiments, the biosyntheticpathway is a BDO biosynthetic pathway.

In addition to the metabolism of methanol, assimilation of formaldehyde,and biosynthesis of BDO, as described herein, the NNOMOs and methodsprovided also can be utilized in various combinations with each otherand with other microbial organisms and methods well known in the art toachieve product biosynthesis by other routes. For example, onealternative to produce BDO, other than use of the BDO producers isthrough addition of another microbial organism capable of converting aBDOP intermediate to BDO. One such procedure includes, for example, thefermentation of a microbial organism that produces a BDOP intermediate.The BDOP intermediate can then be used as a substrate for a secondmicrobial organism that converts the BDOP intermediate to BDO. The BDOPintermediate can be added directly to another culture of the secondorganism or the original culture of the BDOP intermediate producers canbe depleted of these microbial organisms by, for example, cellseparation, and then subsequent addition of the second organism to thefermentation broth can be utilized to produce the final product withoutintermediate purification steps. The same holds true for the MMPs andFAPs provided herein.

In other embodiments, the NNOMOs and methods provided herein can beassembled in a wide variety of subpathways to achieve biosynthesis of,for example, BDO. In these embodiments, biosynthetic pathways for adesired product can be segregated into different microbial organisms,and the different microbial organisms can be co-cultured to produce thefinal product. In such a biosynthetic scheme, the product of onemicrobial organism is the substrate for a second microbial organismuntil the final product is synthesized. For example, the biosynthesis ofBDO can be accomplished by constructing a microbial organism thatcontains biosynthetic pathways for conversion of one pathwayintermediate to another pathway intermediate or the product.Alternatively, BDO also can be biosynthetically produced from microbialorganisms through co-culture or co-fermentation using two organisms inthe same vessel, where the first microbial organism produces a BDOintermediate and the second microbial organism converts the intermediateto BDO. The same holds true for the MMPs and FAPs provided herein.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the NNOMOs and methods together with other microbialorganisms, with the co-culture of other NNOMOs having subpathways andwith combinations of other chemical and/or biochemical procedures wellknown in the art to produce BDO and/or metabolize methanol.

Sources of encoding nucleic acids for a BDO, formaldehyde assimilation,or MMP enzyme or protein can include, for example, any species where theencoded gene product is capable of catalyzing the referenced reaction.Such species include both prokaryotic and eukaryotic organismsincluding, but not limited to, bacteria, including archaea andeubacteria, and eukaryotes, including yeast, plant, insect, animal, andmammal, including human. Exemplary species for such sources include, forexample, Escherichia coli, Saccharomyces cerevisiae, Saccharomyceskluyveri, Candida boidinii, Clostridium kluyveri, Clostridiumacetobutylicum, Clostridium beijerinckii, Clostridiumsaccharoperbutylacetonicum, Clostridium perfringens, Clostridiumdifficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridiumtetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridiumaminobutyricum, Clostridium subterminale, Clostridium sticklandii,Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis,Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus,Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonasputida, Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens,Oryctolagus cuniculus, Rhodobacter spaeroides, Thermoanaerobacterbrockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexusaurantiacus, Roseiflexus castenholzii, Erythrobacter, Simmondsiachinensis, Acinetobacter species, including Acinetobacter calcoaceticusand Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodaii,Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis,Bacillus cereus, Bacillus megaterium, Bacillus brevis, Bacillus pumilus,Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglenagracilis, Treponema denticola, Moorella thermoacetica, Thermotogamaritima, Halobacterium salinarum, Geobacillus stearothermophilus,Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacteriumglutamicum, Acidaminococcus fermentans, Lactococcus lactis,Lactobacillus plantarum, Streptococcus thermophilus, Enterobacteraerogenes, Candida, Aspergillus terreus, Pedicoccus pentosaceus,Zymomonas mobilus, Acetobacter pasteurians, Kluyveromyces lactis,Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis,Natranaerobius thermophilusm, Campylobacter jejuni, Haemophilusinfluenzae, Serratia marcescens, Citrobacter amalonaticus, Myxococcusxanthus, Fusobacterium nuleatum, Penicillium chrysogenum, marine gammaproteobacterium, butyrate-producing bacterium, Nocardia iowensis,Nocardia farcinica, Streptomyces griseus, Schizosaccharomyces pombe,Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio cholera,Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferaxmediterranei, Agrobacterium tumefaciens, Achromobacter denitrificans,Fusobacterium nucleatum, Streptomyces clavuligenus, Acinetobacterbaumanii, Mus musculus, Lachancea kluyveri, Trichomonas vaginalis,Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum,Mesorhizobium loti, Bos taurus, Nicotiana glutinosa, Vibrio vulnificus,Selenomonas ruminantium, Vibrio parahaemolyticus, Archaeoglobusfulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Mycobacteriumsmegmatis MC2 155, Mycobacterium avium subsp. paratuberculosis K-10,Mycobacterium marinum M, Tsukamurella paurometabola DSM 20162, CyanobiumPCC7001, Dictyostelium discoideum AX4, as well as other exemplaryspecies disclosed herein or available as source organisms forcorresponding genes.

In certain embodiments, sources of encoding nucleic acids for a BDO,formaldehyde assimilation, or MMP enzyme or protein includeAcinetobacter baumannii Naval-82, Acinetobacter sp. ADP 1, Acinetobactersp. strain M-1, Actinobacillus succinogenes 130Z, Allochromatium vinosumDSM 180, Amycolatopsis methanolica, Arabidopsis thaliana, Atopobiumparvulum DSM 20469, Azotobacter vinelandii DJ, Bacillus alcalophilusATCC 27647, Bacillus azotoformans LMG 9581, Bacillus coagulans 36D1,Bacillus megaterium, Bacillus methanolicus MGA3, Bacillus methanolicusPB1, Bacillus methanolicus PB-1, Bacillus selenitireducens MLS10,Bacillus smithii, Bacillus subtilis, Burkholderia cenocepacia,Burkholderia cepacia, Burkholderia multivorans, Burkholderia pyrrocinia,Burkholderia stabilis, Burkholderia thailandensis E264, Burkholderialesbacterium Joshi_001, Butyrate-producing bacterium L2-50, Campylobacterjejuni, Candida albicans, Candida boidinii, Candida methylica,Carboxydothermus hydrogenoformans, Carboxydothermus hydrogenoformansZ-2901, Caulobacter sp. AP07, Chloroflexus aggregans DSM 9485,Chloroflexus aurantiacus J-10-fl, Citrobacter freundii, Citrobacterkoseri ATCC BAA-895, Citrobacter youngae, Clostridium, Clostridiumacetobutylicum, Clostridium acetobutylicum ATCC 824, Clostridiumacidurici, Clostridium aminobutyricum, Clostridium asparagiforme DSM15981, Clostridium beijerinckii, Clostridium beijerinckii NCIMB 8052,Clostridium bolteae ATCC BAA-613, Clostridium carboxidivorans P7,Clostridium cellulovorans 743B, Clostridium difficile, Clostridiumhiranonis DSM 13275, Clostridium hylemonae DSM 15053, Clostridiumkluyveri, Clostridium kluyveri DSM 555, Clostridium ljungdahli,Clostridium ljungdahlii DSM 13528, Clostridium methylpentosum DSM 5476,Clostridium pasteurianum, Clostridium pasteurianum DSM 525, Clostridiumperfringens, Clostridium perfringens ATCC 13124, Clostridium perfringensstr. 13, Clostridium phytofermentans ISDg, Clostridiumsaccharobutylicum, Clostridium saccharoperbutylacetonicum, Clostridiumsaccharoperbutylacetonicum N1-4, Clostridium tetani, Corynebacteriumglutamicum ATCC 14067, Corynebacterium glutamicum R, Corynebacterium sp.U-96, Corynebacterium variabile, Cupriavidus necator N-1, CyanobiumPCC7001, Desulfatibacillum alkenivorans AK-01, Desulfitobacteriumhafniense, Desulfitobacterium metallireducens DSM 15288,Desulfotomaculum reducens MI-1, Desulfovibrio africanus str. Walvis Bay,Desulfovibrio fructosovorans JJ, Desulfovibrio vulgaris str.Hildenborough, Desulfovibrio vulgaris str. Miyazaki F, Dictyosteliumdiscoideum AX4, Escherichia coli, Escherichia coli K-12, Escherichiacoli K-12 MG1655, Eubacterium hallii DSM 3353, Flavobacterium frigoris,Fusobacterium nucleatum subsp. polymorphum ATCC 10953, Geobacillus sp.Y4. IMC1, Geobacillus themodenitrificans NG80-2, Geobacter bemidjiensisBem, Geobacter sulfurreducens, Geobacter sulfurreducens PCA, Geobacillusstearothermophilus DSM 2334, Haemophilus influenzae, Helicobacterpylori, Homo sapiens, Hydrogenobacter thermophilus, Hydrogenobacterthermophilus TK-6, Hyphomicrobium denitrificans ATCC 51888,Hyphomicrobium zavarzinii, Klebsiella pneumoniae, Klebsiella pneumoniaesubsp. pneumoniae MGH 78578, Lactobacillus brevis ATCC 367, Leuconostocmesenteroides, Lysinibacillus fusiformis, Lysinibacillus sphaericus,Mesorhizobium loti MAFF303099, Metallosphaera sedula, Methanosarcinaacetivorans, Methanosarcina acetivorans C2A, Methanosarcina barkeri,Methanosarcina mazei Tuc01, Methylobacter marinus, Methylobacteriumextorquens, Methylobacterium extorquens AM1, Methylococcus capsulatas,Methylomonas aminofaciens, Moorella thermoacetica, Mycobacter sp. strainJC1 DSM 3803, Mycobacterium avium subsp. paratuberculosis K-10,Mycobacterium bovis BCG, Mycobacterium gastri, Mycobacterium marinum M,Mycobacterium smegmatis, Mycobacterium smegmatis MC2 155, Mycobacteriumtuberculosis, Nitrosopumilus salaria BD31, Nitrososphaera gargensisGa9.2, Nocardia farcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646),Nostoc sp. PCC 7120, Ogataea angusta, Ogataea parapolymorpha DL-1(Hansenula polymorpha DL-1), Paenibacillus peoriae KCTC 3763, Paracoccusdenitrificans, Penicillium chrysogenum, Photobacterium profundum 3TCK,Phytofermentans ISDg, Pichia pastoris, Picrophilus torridus DSM9790,Porphyromonas gingivalis, Porphyromonas gingivalis W83, Pseudomonasaeruginosa PA01, Pseudomonas denitrificans, Pseudomonas knackmussii,Pseudomonas putida, Pseudomonas sp, Pseudomonas syringae pv. syringaeB728a, Pyrobaculum islandicum DSM 4184, Pyrococcus abyssi, Pyrococcusfuriosus, Pyrococcus horikoshii OT3, Ralstonia eutropha, Ralstoniaeutropha H16, Rhodobacter capsulatus, Rhodobacter sphaeroides,Rhodobacter sphaeroides ATCC 17025, Rhodopseudomonas palustris,Rhodopseudomonas palustris CGA009, Rhodopseudomonas palustris DX-1,Rhodospirillum rubrum, Rhodospirillum rubrum ATCC 11170, Ruminococcusobeum ATCC 29174, Saccharomyces cerevisiae, Saccharomyces cerevisiaeS288c, Salmonella enterica, Salmonella enterica subsp. enterica serovarTyphimurium str. LT2, Salmonella enterica typhimurium, Salmonellatyphimurium, Schizosaccharomyces pombe, Sebaldella termitidis ATCC33386, Shewanella oneidensis MR-1, Sinorhizobium meliloti 1021,Streptomyces coelicolor, Streptomyces griseus subsp. griseus NBRC 13350,Sulfolobus acidocalarius, Sulfolobus solfataricus P-2, Synechocystisstr. PCC 6803, Syntrophobacter fumaroxidans, Thauera aromatica,Thermoanaerobacter sp. X514, Thermococcus kodakaraensis, Thermococcuslitoralis, Thermoplasma acidophilum, Thermoproteus neutrophilus,Thermotoga maritima, Thiocapsa roseopersicina, Tolumonas auensis DSM9187, Trichomonas vaginalis G3, Trypanosoma brucei, Tsukamurellapaurometabola DSM 20162, Vibrio cholera, Vibrio harveyi ATCC BAA-1116,Xanthobacter autotrophicus Py2, Yersinia intermedia, or Zea mays.

However, with the complete genome sequence available for now more than550 species (with more than half of these available on public databasessuch as the NCBI), including 395 microorganism genomes and a variety ofyeast, fungi, plant, and mammalian genomes, the identification of genesencoding the requisite BDO or 4-HB biosynthetic pathway, methanolmetabolic and/or formaldehyde assimilation activity for one or moregenes in related or distant species, including for example, homologues,orthologs, paralogs and nonorthologous gene displacements of knowngenes, and the interchange of genetic alterations between organisms isroutine and well known in the art. Accordingly, the metabolicalterations allowing biosynthesis of BDO or 4-HB, metabolism of methanoland/or assimilation of formaldehyde described herein with reference to aparticular organism such as E. coli can be readily applied to othermicroorganisms, including prokaryotic and eukaryotic organisms alike.Given the teachings and guidance provided herein, those skilled in theart will know that a metabolic alteration exemplified in one organismcan be applied equally to other organisms.

In some instances, such as when an alternative BDO biosynthetic,formaldehyde assimilation and/or MMP exists in an unrelated species, BDObiosynthesis, formaldehyde assimilation and/or methanol metabolism canbe conferred onto the host species by, for example, exogenous expressionof a paralog or paralogs from the unrelated species that catalyzes asimilar, yet non-identical metabolic reaction to replace the referencedreaction. Because certain differences among metabolic networks existbetween different organisms, those skilled in the art will understandthat the actual gene usage between different organisms may differ.However, given the teachings and guidance provided herein, those skilledin the art also will understand that the teachings and methods providedherein can be applied to all microbial organisms using the cognatemetabolic alterations to those exemplified herein to construct amicrobial organism in a species of interest that will synthesize BDO,assimilate formaldehyde and/or metabolize methanol.

Methods for constructing and testing the expression levels of anon-naturally occurring BDO-producing host can be performed, forexample, by recombinant and detection methods well known in the art.Such methods can be found described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); and Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for metabolism ofmethanol, assimilation of formaldehyde and/or production of BDO can beintroduced stably or transiently into a host cell using techniques wellknown in the art including, but not limited to, conjugation,electroporation, chemical transformation, transduction, transfection,and ultrasound transformation. For exogenous expression in E. coli orother prokaryotic cells, some nucleic acid sequences in the genes orcDNAs of eukaryotic nucleic acids can encode targeting signals such asan N-terminal mitochondrial or other targeting signal, which can beremoved before transformation into prokaryotic host cells, if desired.For example, removal of a mitochondrial leader sequence led to increasedexpression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338(2005)). For exogenous expression in yeast or other eukaryotic cells,genes can be expressed in the cytosol without the addition of leadersequence, or can be targeted to mitochondrion or other organelles, ortargeted for secretion, by the addition of a suitable targeting sequencesuch as a mitochondrial targeting or secretion signal suitable for thehost cells. Thus, it is understood that appropriate modifications to anucleic acid sequence to remove or include a targeting sequence can beincorporated into an exogenous nucleic acid sequence to impart desirableproperties. Furthermore, genes can be subjected to codon optimizationwith techniques well known in the art to achieve optimized expression ofthe proteins.

An expression vector or vectors can be constructed to include one ormore BDO biosynthetic, formaldehyde assimilation and/or MMP encodingnucleic acids as exemplified herein operably linked to expressioncontrol sequences functional in the host organism. Expression vectorsapplicable for use in the microbial host organisms provided include, forexample, plasmids, phage vectors, viral vectors, episomes and artificialchromosomes, including vectors and selection sequences or markersoperable for stable integration into a host chromosome. Additionally,the expression vectors can include one or more selectable marker genesand appropriate expression control sequences. Selectable marker genesalso can be included that, for example, provide resistance toantibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture media. Expression controlsequences can include constitutive and inducible promoters,transcription enhancers, transcription terminators, and the like whichare well known in the art. When two or more exogenous encoding nucleicacids are to be co-expressed, both nucleic acids can be inserted, forexample, into a single expression vector or in separate expressionvectors. For single vector expression, the encoding nucleic acids can beoperationally linked to one common expression control sequence or linkedto different expression control sequences, such as one induciblepromoter and one constitutive promoter. The transformation of exogenousnucleic acid sequences involved in a metabolic or synthetic pathway canbe confirmed using methods well known in the art. Such methods include,for example, nucleic acid analysis such as Northern blots or polymerasechain reaction (PCR) amplification of mRNA, or immunoblotting forexpression of gene products, or other suitable analytical methods totest the expression of an introduced nucleic acid sequence or itscorresponding gene product. It is understood by those skilled in the artthat the exogenous nucleic acid is expressed in a sufficient amount toproduce the desired product, and it is further understood thatexpression levels can be optimized to obtain sufficient expression usingmethods well known in the art and as disclosed herein.

Suitable purification and/or assays to test, e.g., for the production ofBDO can be performed using well known methods. Suitable replicates suchas triplicate cultures can be grown for each engineered strain to betested. For example, product and byproduct formation in the engineeredproduction host can be monitored. The final product and intermediates,and other organic compounds, can be analyzed by methods such as HPLC(High Performance Liquid Chromatography), GC-MS (Gas Chromatography-MassSpectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) orother suitable analytical methods using routine procedures well known inthe art. The release of product in the fermentation broth can also betested with the culture supernatant. Byproducts and residual glucose canbe quantified by HPLC using, for example, a refractive index detectorfor glucose and alcohols, and a UV detector for organic acids (Lin etal., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay anddetection methods well known in the art. The individual enzyme orprotein activities from the exogenous DNA sequences can also be assayedusing methods well known in the art. Exemplary assays for the activityof methanol dehydrogenase (FIG. 1, step J) are provided in the ExampleI.

The BDO can be separated from other components in the culture using avariety of methods well known in the art. Such separation methodsinclude, for example, extraction procedures as well as methods thatinclude continuous liquid-liquid extraction, pervaporation, membranefiltration, membrane separation, reverse osmosis, electrodialysis,distillation, crystallization, centrifugation, extractive filtration,ion exchange chromatography, size exclusion chromatography, adsorptionchromatography, and ultrafiltration. All of the above methods are wellknown in the art.

Any of the NNOMOs described herein can be cultured to produce and/orsecrete the biosynthetic products, or intermediates thereof. Forexample, the BDO producers can be cultured for the biosyntheticproduction of BDO. Accordingly, in some embodiments, provided is aculture medium having a BDO, formaldehyde assimilation and/or MMPintermediate described herein. In some aspects, the culture medium canalso be separated from the NNOMOs provided herein that produced the BDO,formaldehyde assimilation and/or MMP intermediate. Methods forseparating a microbial organism from culture medium are well known inthe art. Exemplary methods include filtration, flocculation,precipitation, centrifugation, sedimentation, and the like.

In certain embodiments, for example, for the production of BDO, therecombinant strains are cultured in a medium with carbon source andother essential nutrients. It is sometimes desirable and can be highlydesirable to maintain anaerobic conditions in the fermenter to reducethe cost of the overall process. Such conditions can be obtained, forexample, by first sparging the medium with nitrogen and then sealing theflasks with a septum and crimp-cap. For strains where growth is notobserved anaerobically, microaerobic or substantially anaerobicconditions can be applied by perforating the septum with a small holefor limited aeration. Exemplary anaerobic conditions have been describedpreviously and are well-known in the art. Exemplary aerobic andanaerobic conditions are described, for example, in U.S. Publ. No.2009/0047719. Fermentations can be performed in a batch, fed-batch orcontinuous manner, as disclosed herein. Fermentations can also beconducted in two phases, if desired. The first phase can be aerobic toallow for high growth and therefore high productivity, followed by ananaerobic phase of high BDO yields.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium, can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the NNOMO. Such sources include,for example, sugars, such as glucose, xylose, arabinose, galactose,mannose, fructose, sucrose and starch; or glycerol, alone as the solesource of carbon or in combination with other carbon sources describedherein or known in the art. In one embodiment, the carbon source is asugar. In one embodiment, the carbon source is a sugar-containingbiomass. In some embodiments, the sugar is glucose. In one embodiment,the sugar is xylose. In another embodiment, the sugar is arabinose. Inone embodiment, the sugar is galactose. In another embodiment, the sugaris fructose. In other embodiments, the sugar is sucrose. In oneembodiment, the sugar is starch. In certain embodiments, the carbonsource is glycerol. In some embodiments, the carbon source is crudeglycerol. In one embodiment, the carbon source is crude glycerol withouttreatment. In other embodiments, the carbon source is glycerol andglucose. In another embodiment, the carbon source is methanol andglycerol. In one embodiment, the carbon source is carbon dioxide. In oneembodiment, the carbon source is formate. In one embodiment, the carbonsource is methane. In one embodiment, the carbon source is methanol. Incertain embodiments, methanol is used alone as the sole source of carbonor in combination with other carbon sources described herein or known inthe art. In a specific embodiment, the methanol is the only (sole)carbon source. In one embodiment, the carbon source ischemoelectro-generated carbon (see, e.g., Liao et al. (2012) Science335:1596). In one embodiment, the chemoelectro-generated carbon ismethanol. In one embodiment, the chemoelectro-generated carbon isformate. In one embodiment, the chemoelectro-generated carbon is formateand methanol. In one embodiment, the carbon source is a carbohydrate andmethanol. In one embodiment, the carbon source is a sugar and methanol.In another embodiment, the carbon source is a sugar and glycerol. Inother embodiments, the carbon source is a sugar and crude glycerol. Inyet other embodiments, the carbon source is a sugar and crude glycerolwithout treatment. In one embodiment, the carbon source is asugar-containing biomass and methanol. In another embodiment, the carbonsource is a sugar-containing biomass and glycerol. In other embodiments,the carbon source is a sugar-containing biomass and crude glycerol. Inyet other embodiments, the carbon source is a sugar-containing biomassand crude glycerol without treatment. In some embodiments, the carbonsource is a sugar-containing biomass, methanol and a carbohydrate. Othersources of carbohydrate include, for example, renewable feedstocks andbiomass. Exemplary types of biomasses that can be used as feedstocks inthe methods provided herein include cellulosic biomass, hemicellulosicbiomass and lignin feedstocks or portions of feedstocks. Such biomassfeedstocks contain, for example, carbohydrate substrates useful ascarbon sources such as glucose, xylose, arabinose, galactose, mannose,fructose and starch. Given the teachings and guidance provided herein,those skilled in the art will understand that renewable feedstocks andbiomass other than those exemplified above also can be used forculturing the microbial organisms provided herein for the production ofBDO and other pathway intermediates.

In one embodiment, the carbon source is glycerol. In certainembodiments, the glycerol carbon source is crude glycerol or crudeglycerol without further treatment. In a further embodiment, the carbonsource comprises glycerol or crude glycerol, and also sugar or asugar-containing biomass, such as glucose. In a specific embodiment, theconcentration of glycerol in the fermentation broth is maintained byfeeding crude glycerol, or a mixture of crude glycerol and sugar (e.g.,glucose). In certain embodiments, sugar is provided for sufficientstrain growth. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of glycerol to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 100:1. In one embodiment,the sugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 2:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.In certain other embodiments of the ratios provided above, the glycerolis a crude glycerol or a crude glycerol without further treatment. Inother embodiments of the ratios provided above, the sugar is asugar-containing biomass, and the glycerol is a crude glycerol or acrude glycerol without further treatment.

Crude glycerol can be a by-product produced in the production ofbiodiesel, and can be used for fermentation without any furthertreatment. Biodiesel production methods include (1) a chemical methodwherein the glycerol-group of vegetable oils or animal oils issubstituted by low-carbon alcohols such as methanol or ethanol toproduce a corresponding fatty acid methyl esters or fatty acid ethylesters by transesterification in the presence of acidic or basiccatalysts; (2) a biological method where biological enzymes or cells areused to catalyze transesterification reaction and the correspondingfatty acid methyl esters or fatty acid ethyl esters are produced; and(3) a supercritical method, wherein transesterification reaction iscarried out in a supercritical solvent system without any catalysts. Thechemical composition of crude glycerol can vary with the process used toproduce biodiesel, the transesterification efficiency, recoveryefficiency of the biodiesel, other impurities in the feedstock, andwhether methanol and catalysts were recovered. For example, the chemicalcompositions of eleven crude glycerol collected from seven Australianbiodiesel producers reported that glycerol content ranged between 38%and 96%, with some samples including more than 14% methanol and 29% ash.In certain embodiments, the crude glycerol comprises from 5% to 99%glycerol. In some embodiments, the crude glycerol comprises from 10% to90% glycerol. In some embodiments, the crude glycerol comprises from 10%to 80% glycerol. In some embodiments, the crude glycerol comprises from10% to 70% glycerol. In some embodiments, the crude glycerol comprisesfrom 10% to 60% glycerol. In some embodiments, the crude glycerolcomprises from 10% to 50% glycerol. In some embodiments, the crudeglycerol comprises from 10% to 40% glycerol. In some embodiments, thecrude glycerol comprises from 10% to 30% glycerol. In some embodiments,the crude glycerol comprises from 10% to 20% glycerol. In someembodiments, the crude glycerol comprises from 80% to 90% glycerol. Insome embodiments, the crude glycerol comprises from 70% to 90% glycerol.In some embodiments, the crude glycerol comprises from 60% to 90%glycerol. In some embodiments, the crude glycerol comprises from 50% to90% glycerol. In some embodiments, the crude glycerol comprises from 40%to 90% glycerol. In some embodiments, the crude glycerol comprises from30% to 90% glycerol. In some embodiments, the crude glycerol comprisesfrom 20% to 90% glycerol. In some embodiments, the crude glycerolcomprises from 20% to 40% glycerol. In some embodiments, the crudeglycerol comprises from 40% to 60% glycerol. In some embodiments, thecrude glycerol comprises from 60% to 80% glycerol. In some embodiments,the crude glycerol comprises from 50% to 70% glycerol. In oneembodiment, the glycerol comprises 5% glycerol. In one embodiment, theglycerol comprises 10% glycerol. In one embodiment, the glycerolcomprises 15% glycerol. In one embodiment, the glycerol comprises 20%glycerol. In one embodiment, the glycerol comprises 25% glycerol. In oneembodiment, the glycerol comprises 30% glycerol. In one embodiment, theglycerol comprises 35% glycerol. In one embodiment, the glycerolcomprises 40% glycerol. In one embodiment, the glycerol comprises 45%glycerol. In one embodiment, the glycerol comprises 50% glycerol. In oneembodiment, the glycerol comprises 55% glycerol. In one embodiment, theglycerol comprises 60% glycerol. In one embodiment, the glycerolcomprises 65% glycerol. In one embodiment, the glycerol comprises 70%glycerol. In one embodiment, the glycerol comprises 75% glycerol. In oneembodiment, the glycerol comprises 80% glycerol. In one embodiment, theglycerol comprises 85% glycerol. In one embodiment, the glycerolcomprises 90% glycerol. In one embodiment, the glycerol comprises 95%glycerol. In one embodiment, the glycerol comprises 99% glycerol.

In one embodiment, the carbon source is methanol or formate. In certainembodiments, methanol is used as a carbon source in the FAPs providedherein. In one embodiment, the carbon source is methanol or formate. Inother embodiments, formate is used as a carbon source in the FAPsprovided herein. In specific embodiments, methanol is used as a carbonsource in the MMPs provided herein, either alone or in combination withthe product pathways provided herein. In one embodiment, the carbonsource is methanol. In another embodiment, the carbon source is formate.

In one embodiment, the carbon source comprises methanol, and sugar(e.g., glucose) or a sugar-containing biomass. In another embodiment,the carbon source comprises formate, and sugar (e.g., glucose) or asugar-containing biomass. In one embodiment, the carbon source comprisesmethanol, formate, and sugar (e.g., glucose) or a sugar-containingbiomass. In specific embodiments, the methanol or formate, or both, inthe fermentation feed is provided as a mixture with sugar (e.g.,glucose) or sugar-comprising biomass. In certain embodiments, sugar isprovided for sufficient strain growth.

In certain embodiments, the carbon source comprises methanol and a sugar(e.g., glucose). In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of methanol to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 100:1. In one embodiment,the sugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 2:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises formate and a sugar(e.g., glucose). In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of formate to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 100:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of 2:1. Inone embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises a mixture ofmethanol and formate, and a sugar (e.g., glucose). In certainembodiments, sugar is provided for sufficient strain growth. In someembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of from 200:1 to1:200. In some embodiments, the sugar (e.g., glucose) is provided at amolar concentration ratio of methanol and formate to sugar of from 100:1to 1:100. In some embodiments, the sugar (e.g., glucose) is provided ata molar concentration ratio of methanol and formate to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of methanol and formate to sugar of from50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol and formate to sugarof 100:1. In one embodiment, the sugar (e.g., glucose) is provided at amolar concentration ratio of methanol and formate to sugar of 90:1. Inone embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 80:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 70:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 60:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 50:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 40:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 30:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 20:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 10:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 5:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 2:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:1. In certainembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:100. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:90. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:80. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:70. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:60. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:50. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:40. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:30. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:20. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:10. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:5. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:2. In certainembodiments of the ratios provided above, the sugar is asugar-containing biomass.

Given the teachings and guidance provided herein, those skilled in theart will understand that a NNOMO can be produced that secretes thebiosynthesized compounds when grown on a carbon source such as acarbohydrate. Such compounds include, for example, BDO and any of theintermediate metabolites in the BDOP. All that is required is toengineer in one or more of the required enzyme or protein activities toachieve biosynthesis of the desired compound or intermediate including,for example, inclusion of some or all of the BDO biosynthetic pathways.Accordingly, provided herein is a NNOMO that produces and/or secretesBDO when grown on a carbohydrate or other carbon source and producesand/or secretes any of the intermediate metabolites shown in the BDOPwhen grown on a carbohydrate or other carbon source. The BDO producingmicrobial organisms provided herein can initiate synthesis from anintermediate. The same holds true for intermediates in the formaldehydeassimilation and MMPs.

The NNOMOs provided herein are constructed using methods well known inthe art as exemplified herein to exogenously express at least onenucleic acid encoding a BDO and/or MMP enzyme or protein in sufficientamounts to produce BDO. It is understood that the microbial organismsare cultured under conditions sufficient to produce BDO. Following theteachings and guidance provided herein, the NNOMOs can achievebiosynthesis of BDO, resulting in intracellular concentrations betweenabout 0.1-500 mM or more. Generally, the intracellular concentration ofBDO is between about 3-150 mM, particularly between about 5-125 mM andmore particularly between about 8-100 mM, including about 10 mM, 20 mM,50 mM, 80 mM, or more. Intracellular concentrations between and aboveeach of these exemplary ranges also can be achieved from the NNOMOsprovided herein.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. Publ. No.2009/0047719. Any of these conditions can be employed with the NNOMOs aswell as other anaerobic conditions well known in the art. Under suchanaerobic or substantially anaerobic conditions, the BDO producers cansynthesize BDO at intracellular concentrations of 5-100 mM or more aswell as all other concentrations exemplified herein. It is understoodthat, even though the above description refers to intracellularconcentrations, BDO can produce BDO intracellularly and/or secrete theproduct into the culture medium.

Exemplary fermentation processes include, but are not limited to,fed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation; and continuous fermentation and continuousseparation. In an exemplary batch fermentation protocol, the productionorganism is grown in a suitably sized bioreactor sparged with anappropriate gas. Under anaerobic conditions, the culture is sparged withan inert gas or combination of gases, for example, nitrogen, N2/CO2mixture, argon, helium, and the like. As the cells grow and utilize thecarbon source, additional carbon source(s) and/or other nutrients arefed into the bioreactor at a rate approximately balancing consumption ofthe carbon source and/or nutrients. The temperature of the bioreactor ismaintained at a desired temperature, generally in the range of 22-37degrees C., but the temperature can be maintained at a higher or lowertemperature depending on the growth characteristics of the productionorganism and/or desired conditions for the fermentation process. Growthcontinues for a desired period of time to achieve desiredcharacteristics of the culture in the fermenter, for example, celldensity, product concentration, and the like. In a batch fermentationprocess, the time period for the fermentation is generally in the rangeof several hours to several days, for example, 8 to 24 hours, or 1, 2,3, 4 or 5 days, or up to a week, depending on the desired cultureconditions. The pH can be controlled or not, as desired, in which case aculture in which pH is not controlled will typically decrease to pH 3-6by the end of the run. Upon completion of the cultivation period, thefermenter contents can be passed through a cell separation unit, forexample, a centrifuge, filtration unit, and the like, to remove cellsand cell debris. In the case where the desired product is expressedintracellularly, the cells can be lysed or disrupted enzymatically orchemically prior to or after separation of cells from the fermentationbroth, as desired, in order to release additional product. Thefermentation broth can be transferred to a product separations unit.Isolation of product occurs by standard separations procedures employedin the art to separate a desired product from dilute aqueous solutions.Such methods include, but are not limited to, liquid-liquid extractionusing a water immiscible organic solvent (e.g., toluene or othersuitable solvents, including but not limited to diethyl ether, ethylacetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene,pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether(MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), andthe like) to provide an organic solution of the product, if appropriate,standard distillation methods, and the like, depending on the chemicalcharacteristics of the product of the fermenation process.

In an exemplary fully continuous fermentation protocol, the productionorganism is generally first grown up in batch mode in order to achieve adesired cell density. When the carbon source and/or other nutrients areexhausted, feed medium of the same composition is supplied continuouslyat a desired rate, and fermentation liquid is withdrawn at the samerate. Under such conditions, the product concentration in the bioreactorgenerally remains constant, as well as the cell density. The temperatureof the fermenter is maintained at a desired temperature, as discussedabove. During the continuous fermentation phase, it is generallydesirable to maintain a suitable pH range for optimized production. ThepH can be monitored and maintained using routine methods, including theaddition of suitable acids or bases to maintain a desired pH range. Thebioreactor is operated continuously for extended periods of time,generally at least one week to several weeks and up to one month, orlonger, as appropriate and desired. The fermentation liquid and/orculture is monitored periodically, including sampling up to every day,as desired, to assure consistency of product concentration and/or celldensity. In continuous mode, fermenter contents are constantly removedas new feed medium is supplied. The exit stream, containing cells,medium, and product, are generally subjected to a continuous productseparations procedure, with or without removing cells and cell debris,as desired. Continuous separations methods employed in the art can beused to separate the product from dilute aqueous solutions, includingbut not limited to continuous liquid-liquid extraction using a waterimmiscible organic solvent (e.g., toluene or other suitable solvents,including but not limited to diethyl ether, ethyl acetate,tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane,hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE),dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and thelike), standard continuous distillation methods, and the like, or othermethods well known in the art.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of BDO can includethe addition of an osmoprotectant to the culturing conditions. Incertain embodiments, the NNOMOs provided herein can be sustained,cultured or fermented as described herein in the presence of anosmoprotectant. Briefly, an osmoprotectant refers to a compound thatacts as an osmolyte and helps a microbial organism as described hereinsurvive osmotic stress. Osmoprotectants include, but are not limited to,betaines, amino acids, and the sugar trehalose. Non-limiting examples ofsuch are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate, pipecolicacid, dimethylsulfonioacetate, choline, L-carnitine and ectoine. In oneaspect, the osmoprotectant is glycine betaine. It is understood to oneof ordinary skill in the art that the amount and type of osmoprotectantsuitable for protecting a microbial organism described herein fromosmotic stress will depend on the microbial organism used. The amount ofosmoprotectant in the culturing conditions can be, for example, no morethan about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM,no more than about 1.5 mM, no more than about 2.0 mM, no more than about2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no morethan about 7.0 mM, no more than about 10 mM, no more than about 50 mM,no more than about 100 mM or no more than about 500 mM.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products provided herein can be obtained under anaerobic orsubstantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of BDO, as well as other pathway intermediates, includesanaerobic culture or fermentation conditions. In certain embodiments,the NNOMOs provided can be sustained, cultured or fermented underanaerobic or substantially anaerobic conditions. Briefly, anaerobicconditions refer to an environment devoid of oxygen. Substantiallyanaerobic conditions include, for example, a culture, batch fermentationor continuous fermentation such that the dissolved oxygen concentrationin the medium remains between 0 and 10% of saturation. Substantiallyanaerobic conditions also includes growing or resting cells in liquidmedium or on solid agar inside a sealed chamber maintained with anatmosphere of less than 1% oxygen. The percent of oxygen can bemaintained by, for example, sparging the culture with an N2/CO₂ mixtureor other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of BDO. Exemplary growth proceduresinclude, for example, fed-batch fermentation and batch separation;fed-batch fermentation and continuous separation, or continuousfermentation and continuous separation. All of these processes are wellknown in the art. Fermentation procedures are particularly useful forthe biosynthetic production of commercial quantities of BDO. Generally,and as with non-continuous culture procedures, the continuous and/ornear-continuous production of BDO will include culturing a non-naturallyoccurring BDO-producing organism provided herein in sufficient nutrientsand medium to sustain and/or nearly sustain growth in an exponentialphase. Continuous culture under such conditions can be included, forexample, growth or culturing for 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include longer time periods of 1week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms provided can be cultured for hours, if suitablefor a particular application. It is to be understood that the continuousand/or near-continuous culture conditions also can include all timeintervals in between these exemplary periods. It is further understoodthat the time of culturing the NNOMO provided herein is for a sufficientperiod of time to produce a sufficient amount of product for a desiredpurpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of BDO can be utilized in, for example,fed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation, or continuous fermentation and continuousseparation. Examples of batch and continuous fermentation procedures arewell known in the art.

In addition to the above fermentation procedures using the BDO producersfor continuous production of substantial quantities of BDO, the BDOproducers also can be, for example, simultaneously subjected to chemicalsynthesis procedures to convert the product to other compounds or theproduct can be separated from the fermentation culture and sequentiallysubjected to chemical and/or enzymatic conversion to convert the productto other compounds, if desired.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. Publ. Nos. 2002/0012939, 2003/0224363, 2004/0029149,2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466, and U.S. Pat.No. 7,127,379). Modeling analysis allows reliable predictions of theeffects on cell growth of shifting the metabolism towards more efficientproduction of BDO.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with theNNOMOs for further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. Publ. No. 2002/0168654, International Patent Application No.PCT/US02/00660, and U.S. Publ. No. 2009/0047719.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. Publ. No.2003/0233218, and International Patent Application No. PCT/US03/18838.SimPheny® is a computational system that can be used to produce anetwork model in silico and to simulate the flux of mass, energy orcharge through the chemical reactions of a biological system to define asolution space that contains any and all possible functionalities of thechemical reactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration, somemethods are described herein with reference to the OptKnock computationframework for modeling and simulation. Those skilled in the art willknow how to apply the identification, design and implementation of themetabolic alterations using OptKnock to any of such other metabolicmodeling and simulation computational frameworks and methods well knownin the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. Publ. Nos. 2002/0012939,2003/0224363, 2004/0029149, 2004/0072723, 2003/0059792, 2002/0168654 and2004/0009466, and in U.S. Pat. No. 7,127,379. As disclosed herein, theOptKnock mathematical framework can be applied to pinpoint genedeletions leading to the growth-coupled production of a desired product.Further, the solution of the bilevel OptKnock problem provides only oneset of deletions. To enumerate all meaningful solutions, that is, allsets of knockouts leading to growth-coupled production formation, anoptimization technique, termed integer cuts, can be implemented. Thisentails iteratively solving the OptKnock problem with the incorporationof an additional constraint referred to as an integer cut at eachiteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of aBDOP, FAP, and/or MMP can be introduced into a host organism. In somecases, it can be desirable to modify an activity of a BDO, formaldehydeassimilation, or MMP enzyme or protein to increase production of BDO,formaldehyde and/or reducing equivalents. For example, known mutationsthat increase the activity of a protein or enzyme can be introduced intoan encoding nucleic acid molecule. Additionally, optimization methodscan be applied to increase the activity of an enzyme or protein and/ordecrease an inhibitory activity, for example, decrease the activity of anegative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng. 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng. 22:1-9 (2005); and Sen et al., ApplBiochem. Biotechnol 143:212-223 (2007)) to be effective at creatingdiverse variant libraries, and these methods have been successfullyapplied to the improvement of a wide range of properties across manyenzyme classes. Enzyme characteristics that have been improved and/oraltered by directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Such methods are well known to those skilled in the art. Any ofthese can be used to alter and/or optimize the activity of a BDOPE orprotein. Such methods include, but are not limited to EpPCR, whichintroduces random point mutations by reducing the fidelity of DNApolymerase in PCR reactions (Pritchard et al., J. Theor. Biol.234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA),which is similar to epPCR except a whole circular plasmid is used as thetemplate and random 6-mers with exonuclease resistant thiophosphatelinkages on the last 2 nucleotides are used to amplify the plasmidfollowed by transformation into cells in which the plasmid isre-circularized at tandem repeats (Fujii et al., Nucleic Acids Res.32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)); DNAor Family Shuffling, which typically involves digestion of two or morevariant genes with nucleases such as Dnase I or EndoV to generate a poolof random fragments that are reassembled by cycles of annealing andextension in the presence of DNA polymerase to create a library ofchimeric genes (Stemmer, Proc. Natl. Acad. Sci. U.S.A. 91:10747-10751(1994); and Stemmer, Nature 370:389-391 (1994)); Staggered Extension(StEP), which entails template priming followed by repeated cycles of 2step PCR with denaturation and very short duration ofannealing/extension (as short as 5 sec) (Zhao et al., Nat. Biotechnol.16:258-261 (1998)); Random Priming Recombination (RPR), in which randomsequence primers are used to generate many short DNA fragmentscomplementary to different segments of the template (Shao et al.,Nucleic Acids Res. 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in whichlinearized plasmid DNA is used to form heteroduplexes that are repairedby mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); andVolkov et al., Methods Enzymol. 328:456-463 (2000)); RandomChimeragenesis on Transient Templates (RACHITT), which employs Dnase Ifragmentation and size fractionation of single stranded DNA (ssDNA)(Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extensionon Truncated templates (RETT), which entails template switching ofunidirectionally growing strands from primers in the presence ofunidirectional ssDNA fragments used as a pool of templates (Lee et al.,J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide GeneShuffling (DOGS), in which degenerate primers are used to controlrecombination between molecules; (Bergquist and Gibbs, Methods Mol.Biol. 352:191-204 (2007); Bergquist et al., Biomol. Eng. 22:63-72(2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation forthe Creation of Hybrid Enzymes (ITCHY), which creates a combinatoriallibrary with 1 base pair deletions of a gene or gene fragment ofinterest (Ostermeier et al., Proc. Natl. Acad. Sci. U.S.A. 96:3562-3567(1999); and Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999));Thio-Incremental Truncation for the Creation of Hybrid Enzymes(THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPsare used to generate truncations (Lutz et al., Nucleic Acids Res. 29:E16(2001)); SCRATCHY, which combines two methods for recombining genes,ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. U.S.A.98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in whichmutations made via epPCR are followed by screening/selection for thoseretaining usable activity (Bergquist et al., Biomol. Eng. 22:63-72(2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesismethod that generates a pool of random length fragments using randomincorporation of a phosphothioate nucleotide and cleavage, which is usedas a template to extend in the presence of “universal” bases such asinosine, and replication of an inosine-containing complement givesrandom base incorporation and, consequently, mutagenesis (Wong et al.,Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26(2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)); SyntheticShuffling, which uses overlapping oligonucleotides designed to encode“all genetic diversity in targets” and allows a very high diversity forthe shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255(2002)); Nucleotide Exchange and Excision Technology NexT, whichexploits a combination of dUTP incorporation followed by treatment withuracil DNA glycosylase and then piperidine to perform endpoint DNAfragmentation (Muller et al., Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent ProteinRecombination (SHIPREC), in which a linker is used to facilitate fusionbetween two distantly related or unrelated genes, and a range ofchimeras is generated between the two genes, resulting in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which thestarting materials include a supercoiled double stranded DNA (dsDNA)plasmid containing an insert and two primers which are degenerate at thedesired site of mutations (Kretz et al., Methods Enzymol. 388:3-11(2004)); Combinatorial Cassette Mutagenesis (CCM), which involves theuse of short oligonucleotide cassettes to replace limited regions with alarge number of possible amino acid sequence alterations (Reidhaar-Olsonet al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al.Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis(CMCM), which is essentially similar to CCM and uses epPCR at highmutation rate to identify hot spots and hot regions and then extensionby CMCM to cover a defined region of protein sequence space (Reetz etal., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the MutatorStrains technique, in which conditional is mutator plasmids, utilizingthe mutD5 gene, which encodes a mutant subunit of DNA polymerase III, toallow increases of 20 to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM),which is a multidimensional mutagenesis method that assesses andoptimizes combinatorial mutations of selected amino acids (Rajpal etal., Proc. Natl. Acad. Sci. U.S.A. 102:8466-8471 (2005)); GeneReassembly, which is a DNA shuffling method that can be applied tomultiple genes at one time or to create a large library of chimeras(multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™)Technology supplied by Verenium Corporation), in Silico Protein DesignAutomation (PDA), which is an optimization algorithm that anchors thestructurally defined protein backbone possessing a particular fold, andsearches sequence space for amino acid substitutions that can stabilizethe fold and overall protein energetics, and generally works mosteffectively on proteins with known three-dimensional structures (Hayeset al., Proc. Natl. Acad. Sci. U.S.A. 99:15926-15931 (2002)); andIterative Saturation Mutagenesis (ISM), which involves using knowledgeof structure/function to choose a likely site for enzyme improvement,performing saturation mutagenesis at chosen site using a mutagenesismethod such as Stratagene QUIKCHANGE (Stratagene; San Diego Calif.),screening/selecting for desired properties, and, using improvedclone(s), starting over at another site and continue repeating until adesired activity is achieved (Reetz et al., Nat. Protoc. 2:891-903(2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751(2006)).

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

BDO (or 4-HB) can be harvested or isolated at any time point during theculturing of the microbial organism, for example, in a continuous and/ornear-continuous culture period, as disclosed herein. Generally, thelonger the microorganisms are maintained in a continuous and/ornear-continuous growth phase, the proportionally greater amount of BDOcan be produced.

Therefore, additionally provided is a method for producing BDO thatincludes culturing a non-naturally occurring microbial organism havingone or more gene disruptions, as disclosed herein. The disruptions canoccur in one or more genes encoding an enzyme that increases productionof BDO, including optionally coupling BDO production to growth of themicroorganism when the gene disruption reduces or eliminates an activityof the enzyme. For example, the disruptions can confer stablegrowth-coupled production of BDO onto the non-naturally microbialorganism.

In some embodiments, the gene disruption can include a complete genedeletion. In some embodiments other methods to disrupt a gene include,for example, frameshifting by omission or addition of oligonucleotidesor by mutations that render the gene inoperable. One skilled in the artwill recognize the advantages of gene deletions, however, because of thestability it confers to the non-naturally occurring organism fromreverting to a parental phenotype in which the gene disruption has notoccurred. In particular, the gene disruptions are selected from the genesets as disclosed herein.

Once computational predictions are made of gene sets for disruption toincrease production of BDO, the strains can be constructed, evolved, andtested. Gene disruptions, including gene deletions, are introduced intohost organism by methods well known in the art. A particularly usefulmethod for gene disruption is by homologous recombination, as disclosedherein.

The engineered strains can be characterized by measuring the growthrate, the substrate uptake rate, and/or the product/byproduct secretionrate. Cultures can be grown and used as inoculum for a fresh batchculture for which measurements are taken during exponential growth. Thegrowth rate can be determined by measuring optical density using aspectrophotometer (A600). Concentrations of glucose and other organicacid byproducts in the culture supernatant can be determined by wellknown methods such as HPLC, GC-MS or other well known analytical methodssuitable for the analysis of the desired product, as disclosed herein,and used to calculate uptake and secretion rates.

Strains containing gene disruptions can exhibit suboptimal growth ratesuntil their metabolic networks have adjusted to their missingfunctionalities. To assist in this adjustment, the strains can beadaptively evolved. By subjecting the strains to adaptive evolution,cellular growth rate becomes the primary selection pressure and themutant cells are compelled to reallocate their metabolic fluxes in orderto enhance their rates of growth. This reprogramming of metabolism hasbeen recently demonstrated for several E. coli mutants that had beenadaptively evolved on various substrates to reach the growth ratespredicted a priori by an in silico model (Fong and Palsson, Nat. Genet.36:1056-1058 (2004)). The growth improvements brought about by adaptiveevolution can be accompanied by enhanced rates of BDO production. Thestrains are generally adaptively evolved in replicate, running inparallel, to account for differences in the evolutionary patterns thatcan be exhibited by a host organism (Fong and Palsson, Nat. Genet.36:1056-1058 (2004); Fong et al., J. Bacteriol. 185:6400-6408 (2003);Ibarra et al., Nature 420:186-189 (2002)) that could potentially resultin one strain having superior production qualities over the others.Evolutions can be run for a period of time, typically 2-6 weeks,depending upon the rate of growth improvement attained. In general,evolutions are stopped once a stable phenotype is obtained.

Following the adaptive evolution process, the new strains arecharacterized again by measuring the growth rate, the substrate uptakerate, and the product/byproduct secretion rate. These results arecompared to the theoretical predictions by plotting actual growth andproduction yields alongside the production envelopes from metabolicmodeling. The most successful design/evolution combinations are chosento pursue further, and are characterized in lab-scale batch andcontinuous fermentations. The growth-coupled biochemical productionconcept behind the methods disclosed herein such as OptKnock approachshould also result in the generation of genetically stableoverproducers. Thus, the cultures are maintained in continuous mode foran extended period of time, for example, one month or more, to evaluatelong-term stability. Periodic samples can be taken to ensure that yieldand productivity are maintained.

Adaptive evolution is a powerful technique that can be used to increasegrowth rates of mutant or engineered microbial strains, or of wild-typestrains growing under unnatural environmental conditions. It isespecially useful for strains designed via methods such as OptKnock,which results in growth-coupled product formation. Therefore, evolutiontoward optimal growing strains will indirectly optimize production aswell. Unique strains of E. coli K-12 MG1655 were created through geneknockouts and adaptive evolution. (Fong and Palsson, Nat. Genet.36:1056-1058 (2004)). In this work, all adaptive evolutionary cultureswere maintained in prolonged exponential growth by serial passage ofbatch cultures into fresh medium before the stationary phase wasreached, thus rendering growth rate as the primary selection pressure.Knockout strains were constructed and evolved on minimal mediumsupplemented with different carbon substrates (four for each knockoutstrain). Evolution cultures were carried out in duplicate or triplicate,giving a total of 50 evolution knockout strains. The evolution cultureswere maintained in exponential growth until a stable growth rate wasreached. The computational predictions were accurate (within 10%) atpredicting the post-evolution growth rate of the knockout strains in 38out of the 50 cases examined. Furthermore, a combination of OptKnockdesign with adaptive evolution has led to improved lactic acidproduction strains. (Fong et al., Biotechnol. Bioeng. 91:643-648(2005)). Similar methods can be applied to the strains disclosed hereinand applied to various host strains.

There are a number of developed technologies for carrying out adaptiveevolution. Exemplary methods are disclosed herein. In some embodiments,optimization of a NNOMOs provided herein includes utilizing adaptiveevolution techniques to increase BDO production and/or stability of theproducing strain.

Serial culture involves repetitive transfer of a small volume of grownculture to a much larger vessel containing fresh growth medium. When thecultured organisms have grown to saturation in the new vessel, theprocess is repeated. This method has been used to achieve the longestdemonstrations of sustained culture in the literature (Lenski andTravisano, Proc. Natl. Acad. Sci. USA 91:6808-6814 (1994)) inexperiments which clearly demonstrated consistent improvement inreproductive rate over a period of years. Typically, transfer ofcultures is usually performed during exponential phase, so each day thetransfer volume is precisely calculated to maintain exponential growththrough the next 24 hour period. Manual serial dilution is inexpensiveand easy to parallelize.

In continuous culture the growth of cells in a chemostat represents anextreme case of dilution in which a very high fraction of the cellpopulation remains. As a culture grows and becomes saturated, a smallproportion of the grown culture is replaced with fresh media, allowingthe culture to continually grow at close to its maximum population size.Chemostats have been used to demonstrate short periods of rapidimprovement in reproductive rate (Dykhuizen, Methods Enzymol. 613-631(1993)). The potential usefulness of these devices was recognized, buttraditional chemostats were unable to sustain long periods of selectionfor increased reproduction rate, due to the unintended selection ofdilution-resistant (static) variants. These variants are able to resistdilution by adhering to the surface of the chemostat, and by doing so,outcompete less adherent individuals, including those that have higherreproductive rates, thus obviating the intended purpose of the device(Chao and Ramsdell, J. Gen. Microbiol 20:132-138 (1985)). One possibleway to overcome this drawback is the implementation of a device with twogrowth chambers, which periodically undergo transient phases ofsterilization, as described previously (Marliere and Mutzel, U.S. Pat.No. 6,686,194).

Evolugator™ is a continuous culture device developed by Evolugate, LLC(Gainesville, Fla.) and exhibits significant time and effort savingsover traditional evolution techniques (de Crecy et al., Appl. Microbiol.Biotechnol. 77:489-496 (2007)). The cells are maintained in prolongedexponential growth by the serial passage of batch cultures into freshmedium before the stationary phase is attained. By automating opticaldensity measurement and liquid handling, the Evolugator™ can performserial transfer at high rates using large culture volumes, thusapproaching the efficiency of a chemostat in evolution of cell fitness.For example, a mutant of Acinetobacter sp ADP1 deficient in a componentof the translation apparatus, and having severely hampered growth, wasevolved in 200 generations to 80% of the wild-type growth rate. However,in contrast to the chemostat which maintains cells in a single vessel,the machine operates by moving from one “reactor” to the next insubdivided regions of a spool of tubing, thus eliminating any selectionfor wall-growth. The transfer volume is adjustable, and normally set toabout 50%. A drawback to this device is that it is large and costly,thus running large numbers of evolutions in parallel is not practical.Furthermore, gas addition is not well regulated, and strict anaerobicconditions are not maintained with the current device configuration.Nevertheless, this is an alternative method to adaptively evolve aproduction strain.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

4. EXAMPLES 4.1 Example I—Production of Reducing Equivalents Via a MMP

Exemplary MMPs are provided in FIG. 1.

FIG. 1, Step A—Methanol Methyltransferase (EM1)

A complex of 3-methyltransferase proteins, denoted MtaA, MtaB, and MtaC,perform the desired EM1 activity (Sauer et al., Eur. J. Biochem.243:670-677 (1997); Naidu and Ragsdale, J. Bacteriol. 183:3276-3281(2001); Tallant and Krzycki, J. Biol. Chem. 276:4485-4493 (2001);Tallant and Krzycki, J. Bacteriol. 179:6902-6911 (1997); Tallant andKrzycki, J. Bacteriol. 178:1295-1301 (1996); Ragsdale, S. W., Crit. Rev.Biochem. Mol. Biol. 39:165-195 (2004)).

MtaB is a zinc protein that can catalyze the transfer of a methyl groupfrom methanol to MtaC, a corrinoid protein. Exemplary genes encodingMtaB and MtaC can be found in methanogenic archaea such asMethanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931(2006) and Methanosarcina acetivorans (Galagan et al., Genome Res.12:532-542 (2002), as well as the acetogen, Moorella thermoacetica (Daset al., Proteins 67:167-176 (2007). In general, the MtaB and MtaC genesare adjacent to one another on the chromosome as their activities aretightly interdependent. The protein sequences of various MtaB and MtaCencoding genes in M. barkeri, M. acetivorans, and M. thermoaceticum canbe identified by their following GenBank accession numbers.

Protein GenBank ID GI number Organism MtaB1 YP_304299 73668284Methanosarcina barkeri MtaC1 YP_304298 73668283 Methanosarcina barkeriMtaB2 YP_307082 73671067 Methanosarcina barkeri MtaC2 YP_307081 73671066Methanosarcina barkeri MtaB3 YP_304612 73668597 Methanosarcina barkeriMtaC3 YP_304611 73668596 Methanosarcina barkeri MtaB1 NP_615421 20089346Methanosarcina acetivorans MtaB1 NP_615422 20089347 Methanosarcinaacetivorans MtaB2 NP_619254 20093179 Methanosarcina acetivorans MtaC2NP_619253 20093178 Methanosarcina acetivorans MtaB3 NP_616549 20090474Methanosarcina acetivorans MtaC3 NP_616550 20090475 Methanosarcinaacetivorans MtaB YP_430066 83590057 Moorella thermoacetica MtaCYP_430065 83590056 Moorella thermoacetica MtaA YP_430064 83590056Moorella thermoacetica

The MtaB1 and MtaC1 genes, YP_304299 and YP_304298, from M. barkeri werecloned into E. coli and sequenced (Sauer et al., Eur. J. Biochem.243:670-677 (1997)). The crystal structure of this methanol-cobalaminmethyltransferase complex is also available (Hagemeier et al., Proc.Natl. Acad. Sci. U.S.A. 103:18917-18922 (2006)). The MtaB genes,YP_307082 and YP_304612, in M. barkeri were identified by sequencehomology to YP_304299. In general, homology searches are an effectivemeans of identifying EM1s because MtaB encoding genes show little or nosimilarity to methyltransferases that act on alternative substrates suchas trimethylamine, dimethylamine, monomethylamine, or dimethylsulfide.The MtaC genes, YP_307081 and YP_304611 were identified based on theirproximity to the MtaB genes and also their homology to YP_304298. Thethree sets of MtaB and MtaC genes from M. acetivorans have beengenetically, physiologically, and biochemically characterized (Pritchettand Metcalf, Mol. Microbiol. 56:1183-1194 (2005)). Mutant strainslacking two of the sets were able to grow on methanol, whereas a strainlacking all three sets of MtaB and MtaC genes sets could not grow onmethanol. This suggests that each set of genes plays a role in methanolutilization. The M. thermoacetica MtaB gene was identified based onhomology to the methanogenic MtaB genes and also by its adjacentchromosomal proximity to the methanol-induced corrinoid protein, MtaC,which has been crystallized (Zhou et al., Acta Crystallogr. Sect. F.Struct. Biol. Cyrst. Commun. 61:537-540 (2005) and further characterizedby Northern hybridization and Western Blotting ((Das et al., Proteins67:167-176 (2007)). MtaA is zinc protein that catalyzes the transfer ofthe methyl group from MtaC to either Coenzyme M in methanogens ormethyltetrahydrofolate in acetogens. MtaA can also utilizemethylcobalamin as the methyl donor. Exemplary genes encoding MtaA canbe found in methanogenic archaea such as Methanosarcina barkeri (Maederet al., J. Bacteriol. 188:7922-7931 (2006) and Methanosarcinaacetivorans (Galagan et al., Genome Res. 12:532-542 (2002), as well asthe acetogen, Moorella thermoacetica ((Das et al., Proteins 67:167-176(2007)). In general, MtaA proteins that catalyze the transfer of themethyl group from CH₃-MtaC are difficult to identify bioinformaticallyas they share similarity to other corrinoid protein methyltransferasesand are not oriented adjacent to the MtaB and MtaC genes on thechromosomes. Nevertheless, a number of MtaA encoding genes have beencharacterized. The protein sequences of these genes in M. barkeri and M.acetivorans can be identified by the following GenBank accessionnumbers.

Protein GenBank ID GI number Organism MtaA YP_304602 73668587Methanosarcina barkeri MtaA1 NP_619241 20093166 Methanosarcinaacetivorans MtaA2 NP_616548 20090473 Methanosarcina acetivorans

The MtaA gene, YP_304602, from M. barkeri was cloned, sequenced, andfunctionally overexpressed in E. coli (Harms and Thauer, Eur. J.Biochem. 235:653-659 (1996)). In M. acetivorans, MtaA1 is required forgrowth on methanol, whereas MtaA2 is dispensable even though methaneproduction from methanol is reduced in MtaA2 mutants (Bose et al., J.Bacteriol. 190:4017-4026 (2008)). There are multiple additional MtaAhomologs in M. barkeri and M. acetivorans that are as yetuncharacterized, but may also catalyze corrinoid proteinmethyltransferase activity.

Putative MtaA encoding genes in M. thermoacetica were identified bytheir sequence similarity to the characterized methanogenic MtaA genes.Specifically, three M. thermoacetica genes show high homology (>30%sequence identity) to YP_304602 from M. barkeri. Unlike methanogenicMtaA proteins that naturally catalyze the transfer of the methyl groupfrom CH3-MtaC to Coenzyme M, an M. thermoacetica MtaA is likely totransfer the methyl group to methyltetrahydrofolate given the similarroles of methyltetrahydrofolate and Coenzyme M in methanogens andacetogens, respectively. The protein sequences of putative MtaA encodinggenes from M. thermoacetica can be identified by the following GenBankaccession numbers.

Protein GenBank ID GI number Organism MtaA YP_430937 83590928 Moorellathermoacetica MtaA YP_431175 83591166 Moorella thermoacetica MtaAYP_430935 83590926 Moorella thermoacetica MtaA YP_430064 83590056Moorella thermoacetica

FIG. 1, Step B—Methylenetetrahydrofolate Reductase (EM2)

The conversion of methyl-THF to methylenetetrahydrofolate is catalyzedby EM2. In M. thermoacetica, this enzyme is oxygen-sensitive andcontains an iron-sulfur cluster (Clark and Ljungdahl, J. Biol. Chem.259:10845-10849 (1984). This enzyme is encoded by metF in E. coli(Sheppard et al., J. Bacteriol. 181:718-725 (1999) and CHY 1233 in C.hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005). The M.thermoacetica genes, and its C. hydrogenoformans counterpart, arelocated near the CODH/ACS gene cluster, separated by putativehydrogenase and heterodisulfide reductase genes. Some additional genecandidates found bioinformatically are listed below. In Acetobacteriumwoodii metF is coupled to the Rnf complex through RnfC2 (Poehlein et al,PLoS One. 7:e33439). Homologs of RnfC are found in other organisms byblast search. The Rnf complex is known to be a reversible complex (Fuchs(2011) Annu. Rev. Microbiol. 65:631-658).

Protein GenBank ID GI number Organism Moth_1191 YP_430048.1 83590039Moorella thermoacetica Moth_1192 YP_430049.1 83590040 Moorellathermoacetica metF NP_418376.1 16131779 Escherichia coli CHY_1233YP_360071.1 78044792 Carboxydothermus hydrogenoformans CLJU_c37610YP_003781889.1 300856905 Clostridium ljungdahlii DSM 13528DesfrDRAFT_3717 ZP_07335241.1 303248996 Desulfovibrio fructosovorans JJCcarbDRAFT_2950 ZP_05392950.1 255526026 Clostridium carboxidivorans P7Ccel74_010100023124 ZP_07633513.1 307691067 Clostridium cellulovorans743B Cphy_3110 YP_001560205.1 160881237 Clostridium phytofermentans ISDg

FIG. 1, Steps C and D—Methylenetetrahydrofolate Dehydrogenase (EM3),Methenyltetrahydrofolate Cyclohydrolase (EM4)

In M. thermoacetica, E. coli, and C. hydrogenoformans, EM4 and EM3 arecarried out by the bi-functional gene products of Moth_1516, folD, andCHY_1878, respectively (Pierce et al., Environ. Microbiol. 10:2550-2573(2008); Wu et al., PLoS Genet. 1:e65 (2005); D'Ari and Rabinowitz, J.Biol. Chem. 266:23953-23958 (1991)). A homolog exists in C.carboxidivorans P7. Several other organisms also encode for thisbifunctional protein as tabulated below.

Protein GenBank ID GI number Organism Moth_1516 YP_430368.1 83590359Moorella thermoacetica folD NP_415062.1 16128513 Escherichia coliCHY_1878 YP_360698.1 78044829 Carboxydothermus hydrogenoformansCcarbDRAFT_2948 ZP_05392948.1 255526024 Clostridium carboxidivorans P7folD ADK16789.1 300437022 Clostridium ljungdahlii DSM 13528 folD-2NP_951919.1 39995968 Geobacter sulfurreducens PCA folD YP_725874.1113867385 Ralstonia eutropha H16 folD NP_348702.1 15895353 Clostridiumacetobutylicum ATCC 824 folD YP_696506.1 110800457 Clostridiumperfringens MGA3_09460 EIJ83438.1 387591119 Bacillus methanolicus MGA3PB1_14689 ZP_10132349.1 387929672 Bacillus methanolicus PB1

FIG. 1, Step E—Formyltetrahydrofolate Deformylase (EM5)

This enzyme catalyzes the hydrolysis of 10-formyltetrahydrofolate(formyl-THF) to THF and formate. In E. coli, this enzyme is encoded bypurU and has been overproduced, purified, and characterized (Nagy, etal., J. Bacteriol. 3:1292-1298 (1995)). Homologs exist inCorynebacterium sp. U-96 (Suzuki, et al., Biosci. Biotechnol. Biochem.69(5):952-956 (2005)), Corynebacterium glutamicum ATCC 14067, Salmonellaenterica, and several additional organisms.

Protein GenBank ID GI number Organism purU AAC74314.1 1787483Escherichia coli K-12 MG1655 purU BAD97821.1 63002616 Corynebacteriumsp. U-96 purU EHE84645.1 354511740 Corynebacterium glutamicum ATCC 14067purU NP_460715.1 16765100 Salmonella enterica subsp. enterica serovarTyphimurium str. LT2

FIG. 1, Step F—Formyltetrahydrofolate Synthetase (EM6)

Formyltetrahydrofolate synthetase ligates formate to tetrahydrofolate atthe expense of one ATP. This reaction is catalyzed by the gene productof Moth_0109 in M. thermoacetica (O'brien et al., Experientia Suppl.26:249-262 (1976); Lovell et al., Arch. Microbiol. 149:280-285 (1988);Lovell et al., Biochemistry 29:5687-5694 (1990)), FHS in Clostridiumacidurici (Whitehead and Rabinowitz, J. Bacteriol. 167:203-209 (1986);Whitehead and Rabinowitz, J. Bacteriol. 170:3255-3261 (1988), andCHY_2385 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005).Homologs exist in C. carboxidivorans P7. This enzyme is found in severalother organisms as listed below.

Protein GenBank ID GI number Organism Moth_0109 YP_428991.1 83588982Moorella thermoacetica CHY_2385 YP_361182.1 78045024 Carboxydothermushydrogenoformans FHS P13419.1 120562 Clostridium aciduriciCcarbDRAFT_1913 ZP_05391913.1 255524966 Clostridium carboxidivorans P7CcarbDRAFT_2946 ZP_05392946.1 255526022 Clostridium carboxidivorans P7Dhaf_0555 ACL18622.1 219536883 Desulfitobacterium hafniense fhsYP_001393842.1 153953077 Clostridium kluyveri DSM 555 fhs YP_003781893.1 300856909 Clostridium ljungdahlii DSM 13528 MGA3_08300EIJ83208.1 387590889 Bacillus methanolicus MGA3 PB1_13509 ZP_10132113.1387929436 Bacillus methanolicus PB1

FIG. 1, Step G—Formate Hydrogen Lyase (EM15)

An EM15 enzyme can be employed to convert formate to carbon dioxide andhydrogen. An exemplary EM15 enzyme can be found in Escherichia coli. TheE. coli EM15 consists of hydrogenase 3 and formate dehydrogenase-H(Maeda et al., Appl Microbiol Biotechnol 77:879-890 (2007)). It isactivated by the gene product of fhIA. (Maeda et al., Appl MicrobiolBiotechnol 77:879-890 (2007)). The addition of the trace elements,selenium, nickel and molybdenum, to a fermentation broth has been shownto enhance EM15 activity (Soini et al., Microb. Cell Fact. 7:26 (2008)).Various hydrogenase 3, EM8 and transcriptional activator genes are shownbelow.

Protein GenBank ID GI number Organism hycA NP_417205 16130632Escherichia coli K-12 MG1655 hycB NP_417204 16130631 Escherichia coliK-12 MG1655 hycC NP_417203 16130630 Escherichia coli K-12 MG1655 hycDNP_417202 16130629 Escherichia coli K-12 MG1655 hycE NP_417201 16130628Escherichia coli K-12 MG1655 hycF NP_417200 16130627 Escherichia coliK-12 MG1655 hycG NP_417199 16130626 Escherichia coli K-12 MG1655 hycHNP_417198 16130625 Escherichia coli K-12 MG1655 hycI NP_417197 16130624Escherichia coli K-12 MG1655 fdhF NP_418503 16131905 Escherichia coliK-12 MG1655 fhlA NP_417211 16130638 Escherichia coli K-12 MG1655

An EM15 enzyme also exists in the hyperthermophilic archaeon,Thermococcus litoralis (Takacs et al., BMC. Microbiol 8:88 (2008)).

Protein GenBank ID GI number Organism mhyC ABW05543 157954626 mhyDABW05544 157954627 Thermococcus litoralis mhyE ABW05545 157954628Thermococcus litoralis myhF ABW05546 157954629 Thermococcus litoralismyhG ABW05547 157954630 Thermococcus litoralis myhH ABW05548 157954631Thermococcus litoralis fdhA AAB94932 2746736 Thermococcus litoralis fdhBAAB94931 157954625 Thermococcus litoralis

Additional EM15 systems have been found in Salmonella typhimurium,Klebsiella pneumoniae, Rhodospirillum rubrum, Methanobacteriumformicicum (Vardar-Schara et al., Microbial Biotechnology 1:107-125(2008)).

FIG. 1, Step H—Hydrogenase (EM16)

Hydrogenase enzymes can convert hydrogen gas to protons and transferelectrons to acceptors such as ferredoxins, NAD+, or NADP+. Ralstoniaeutropha H16 uses hydrogen as an energy source with oxygen as a terminalelectron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an“O2-tolerant” EM16 (Cracknell, et al. Proc Nat Acad Sci, 106(49)20681-20686 (2009)) that is periplasmically-oriented and connected tothe respiratory chain via a b-type cytochrome (Schink and Schlegel,Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J.Biochem. 248, 179-186 (1997)). R. eutropha also contains an 02-tolerantsoluble EM16 encoded by the Hox operon which is cytoplasmic and directlyreduces NAD+ at the expense of hydrogen (Schneider and Schlegel,Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J. Bact. 187(9)3122-3132(2005)). Soluble EM16 enzymes are additionally present inseveral other organisms including Geobacter sulfurreducens (Coppi,Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803(Germer, J. Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsaroseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728(2004)). The Synechocystis enzyme is capable of generating NADPH fromhydrogen. Overexpression of both the Hox operon from Synechocystis str.PCC 6803 and the accessory genes encoded by the Hyp operon from Nostocsp. PCC 7120 led to increased EM16 activity compared to expression ofthe Hox genes alone (Germer, J. Biol. Chem. 284(52), 36462-36472(2009)).

Protein GenBank ID GI Number Organism HoxF NP_942727.1 38637753Ralstonia eutropha H16 HoxU NP_942728.1 38637754 Ralstonia eutropha H16HoxY NP_942729.1 38637755 Ralstonia eutropha H16 HoxH NP_942730.138637756 Ralstonia eutropha H16 HoxW NP_942731.1 38637757 Ralstoniaeutropha H16 HoxI NP_942732.1 38637758 Ralstonia eutropha H16 HoxENP_953767.1 39997816 Geobacter sulfurreducens HoxF NP_953766.1 39997815Geobacter sulfurreducens HoxU NP_953765.1 39997814 Geobactersulfurreducens HoxY NP_953764.1 39997813 Geobacter sulfurreducens HoxHNP_953763.1 39997812 Geobacter sulfurreducens GSU2717 NP_953762.139997811 Geobacter sulfurreducens HoxE NP_441418.1 16330690Synechocystis str. PCC 6803 HoxF NP_441417.1 16330689 Synechocystis str.PCC 6803 Unknown NP_441416.1 16330688 Synechocystis str. PCC 6803function HoxU NP_441415.1 16330687 Synechocystis str. PCC 6803 HoxYNP_441414.1 16330686 Synechocystis str. PCC 6803 Unknown NP_441413.116330685 Synechocystis str. PCC 6803 function Unknown NP_441412.116330684 Synechocystis str. PCC 6803 function HoxH NP_441411.1 16330683Synechocystis str. PCC 6803 HypF NP_484737.1 17228189 Nostoc sp. PCC7120 HypC NP_484738.1 17228190 Nostoc sp. PCC 7120 HypD NP_484739.117228191 Nostoc sp. PCC 7120 Unknown NP_484740.1 17228192 Nostoc sp. PCC7120 function HypE NP_484741.1 17228193 Nostoc sp. PCC 7120 HypANP_484742.1 17228194 Nostoc sp. PCC 7120 HypB NP_484743.1 17228195Nostoc sp. PCC 7120 Hox1E AAP50519.1 37787351 Thiocapsa roseopersicinaHox1F AAP50520.1 37787352 Thiocapsa roseopersicina Hox1U AAP50521.137787353 Thiocapsa roseopersicina Hox1Y AAP50522.1 37787354 Thiocapsaroseopersicina Hox1H AAP50523.1 37787355 Thiocapsa roseopersicina

The genomes of E. coli and other enteric bacteria encode up to four EM16enzymes (Sawers, G., Antonie Van Leeuwenhoek 66:57-88 (1994); Sawers etal., J. Bacteriol. 164:1324-1331 (1985); Sawers and Boxer, Eur. J.Biochem. 156:265-275 (1986); Sawers et al., J. Bacteriol. 168:398-404(1986)). Given the multiplicity of enzyme activities E. coli or anotherhost organism can provide sufficient EM16 activity to split incomingmolecular hydrogen and reduce the corresponding acceptor. Endogenoushydrogen-lyase enzymes of E. coli include hydrogenase 3, amembrane-bound enzyme complex using ferredoxin as an acceptor, andhydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4are encoded by the hyc and hyf gene clusters, respectively. EM16activity in E. coli is also dependent upon the expression of the hypgenes whose corresponding proteins are involved in the assembly of theEM16 complexes (Jacobi et al., Arch. Microbiol 158:444-451 (1992);Rangarajan et al., J. Bacteriol. 190:1447-1458 (2008)). The M.thermoacetica and Clostridium ljungdahli EM16s are suitable for a hostthat lacks sufficient endogenous EM16 activity. M. thermoacetica and C.ljungdahli can grow with CO₂ as the exclusive carbon source indicatingthat reducing equivalents are extracted from H2 to enable acetyl-CoAsynthesis via the Wood-Ljungdahl pathway (Drake, H. L., J. Bacteriol.150:702-709 (1982); Drake and Daniel, Res Microbiol 155:869-883 (2004);Kellum and Drake, J. Bacteriol. 160:466-469 (1984)). M. thermoaceticahas homologs to several hyp, hyc, and hyf genes from E. coli. Theseprotein sequences encoded for by these genes are identified by thefollowing GenBank accession numbers. In addition, several gene clustersencoding EM16 functionality are present in M. thermoacetica and C.ljungdahli (see for example US 2012/0003652).

Protein GenBank ID GI Number Organism HypA NP_417206 16130633Escherichia coli HypB NP_417207 16130634 Escherichia coli HypC NP_41720816130635 Escherichia coli HypD NP_417209 16130636 Escherichia coli HypENP_417210 226524740 Escherichia coli HypF NP_417192 16130619 Escherichiacoli HycA NP_417205 16130632 Escherichia coli HycB NP_417204 16130631Escherichia coli HycC NP_417203 16130630 Escherichia coli HycD NP_41720216130629 Escherichia coli HycE NP_417201 16130628 Escherichia coli HycFNP_417200 16130627 Escherichia coli HycG NP_417199 16130626 Escherichiacoli HycH NP_417198 16130625 Escherichia coli HycI NP_417197 16130624Escherichia coli HyfA NP_416976 90111444 Escherichia coli HyfB NP_41697716130407 Escherichia coli HyfC NP_416978 90111445 Escherichia coli HyfDNP_416979 16130409 Escherichia coli HyfE NP_416980 16130410 Escherichiacoli HyfF NP_416981 16130411 Escherichia coli HyfG NP_416982 16130412Escherichia coli HyfH NP_416983 16130413 Escherichia coli HyfI NP_41698416130414 Escherichia coli HyfJ NP_416985 90111446 Escherichia coli HyfRNP_416986 90111447 Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. coliEM16 genes are shown below.

Protein GenBank ID GI Number Organism Moth_2175 YP_431007 83590998Moorella thermoacetica Moth_2176 YP_431008 83590999 Moorellathermoacetica Moth_2177 YP_431009 83591000 Moorella thermoaceticaMoth_2178 YP_431010 83591001 Moorella thermoacetica Moth_2179 YP_43101183591002 Moorella thermoacetica Moth_2180 YP_431012 83591003 Moorellathermoacetica Moth_2181 YP_431013 83591004 Moorella thermoaceticaMoth_2182 YP_431014 83591005 Moorella thermoacetica Moth_2183 YP_43101583591006 Moorella thermoacetica Moth_2184 YP_431016 83591007 Moorellathermoacetica Moth_2185 YP_431017 83591008 Moorella thermoaceticaMoth_2186 YP_431018 83591009 Moorella thermoacetica Moth_2187 YP_43101983591010 Moorella thermoacetica Moth_2188 YP_431020 83591011 Moorellathermoacetica Moth_2189 YP_431021 83591012 Moorella thermoaceticaMoth_2190 YP_431022 83591013 Moorella thermoacetica Moth_2191 YP_43102383591014 Moorella thermoacetica Moth_2192 YP_431024 83591015 Moorellathermoacetica Moth_0439 YP_429313 83589304 Moorella thermoaceticaMoth_0440 YP_429314 83589305 Moorella thermoacetica Moth_0441 YP_42931583589306 Moorella thermoacetica Moth_0442 YP_429316 83589307 Moorellathermoacetica Moth_0809 YP_429670 83589661 Moorella thermoaceticaMoth_0810 YP_429671 83589662 Moorella thermoacetica Moth_0811 YP_42967283589663 Moorella thermoacetica Moth_0812 YP_429673 83589664 Moorellathermoacetica Moth_0814 YP_429674 83589665 Moorella thermoaceticaMoth_0815 YP_429675 83589666 Moorella thermoacetica Moth_0816 YP_42967683589667 Moorella thermoacetica Moth_1193 YP_430050 83590041 Moorellathermoacetica Moth_1194 YP_430051 83590042 Moorella thermoaceticaMoth_1195 YP_430052 83590043 Moorella thermoacetica Moth_1196 YP_43005383590044 Moorella thermoacetica Moth_1717 YP_430562 83590553 Moorellathermoacetica Moth_1718 YP_430563 83590554 Moorella thermoaceticaMoth_1719 YP_430564 83590555 Moorella thermoacetica Moth_1883 YP_43072683590717 Moorella thermoacetica Moth_1884 YP_430727 83590718 Moorellathermoacetica Moth_1885 YP_430728 83590719 Moorella thermoaceticaMoth_1886 YP_430729 83590720 Moorella thermoacetica Moth_1887 YP_43073083590721 Moorella thermoacetica Moth_1888 YP_430731 83590722 Moorellathermoacetica Moth_1452 YP_430305 83590296 Moorella thermoaceticaMoth_1453 YP_430306 83590297 Moorella thermoacetica Moth_1454 YP_43030783590298 Moorella thermoacetica

Genes encoding EM16 enzymes from C. ljungdahli are shown below.

Protein GenBank ID GI Number Organism CLJU_c20290 ADK15091.1 300435324Clostridium ljungdahli CLJU_c07030 ADK13773.1 300434006 Clostridiumljungdahli CLJU_c07040 ADK13774.1 300434007 Clostridium ljungdahliCLJU_c07050 ADK13775.1 300434008 Clostridium ljungdahli CLJU_c07060ADK13776.1 300434009 Clostridium ljungdahli CLJU_c07070 ADK13777.1300434010 Clostridium ljungdahli CLJU_c07080 ADK13778.1 300434011Clostridium ljungdahli CLJU_c14730 ADK14541.1 300434774 Clostridiumljungdahli CLJU_c14720 ADK14540.1 300434773 Clostridium ljungdahliCLJU_c14710 ADK14539.1 300434772 Clostridium ljungdahli CLJU_c14700ADK14538.1 300434771 Clostridium ljungdahli CLJU_c28670 ADK15915.1300436148 Clostridium ljungdahli CLJU_c28660 ADK15914.1 300436147Clostridium ljungdahli CLJU_c28650 ADK15913.1 300436146 Clostridiumljungdahli CLJU_c28640 ADK15912.1 300436145 Clostridium ljungdahli

In some cases, EM16 encoding genes are located adjacent to a CODH. InRhodospirillum rubrum, the encoded CODH/hydrogenase proteins form amembrane-bound enzyme complex that has been indicated to be a site whereenergy, in the form of a proton gradient, is generated from theconversion of CO and H₂O to CO₂ and H2 (Fox et al., J Bacteriol.178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and itsadjacent genes have been proposed to catalyze a similar functional rolebased on their similarity to the R. rubrum CODH/hydrogenase gene cluster(Wu et al., PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-Iwas also shown to exhibit intense CO oxidation and CO₂ reductionactivities when linked to an electrode (Parkin et al. J Am. Chem. Soc.129:10328-10329 (20071).

Protein GenBank ID GI Number Organism CooL AAC45118 1515468Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum CooUAAC45120 1515470 Rhodospirillum rubrum CooH AAC45121 1498746Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum rubrum CODH(CooS) AAC45123 1498748 Rhodospirillum rubrum CooC AAC45124 1498749Rhodospirillum rubrum CooT AAC45125 1498750 Rhodospirillum rubrum CooJAAC45126 1498751 Rhodospirillum rubrum CODH-I (CooS-I) YP_36064478043475 Carboxydothermus hydrogenoformans CooF YP_360645 78044791Carboxydothermus hydrogenoformans HypA YP_360646 78044340Carboxydothermus hydrogenoformans CooH YP_360647 78043871Carboxydothermus hydrogenoformans CooU YP_360648 78044023Carboxydothermus hydrogenoformans CooX YP_360649 78043124Carboxydothermus hydrogenoformans CooL YP_360650 78043938Carboxydothermus hydrogenoformans CooK YP_360651 78044700Carboxydothermus hydrogenoformans CooM YP_360652 78043942Carboxydothermus hydrogenoformans CooC YP_360654.1 78043296Carboxydothermus hydrogenoformans CooA-1 YP_360655.1 78044021Carboxydothermus _(—) hydrogenoformans

Some EM16 and CODH enzymes transfer electrons to ferredoxins.Ferredoxins are small acidic proteins containing one or more iron-sulfurclusters that function as intracellular electron carriers with a lowreduction potential. Reduced ferredoxins donate electrons toFe-dependent enzymes such as ferredoxin-NADP⁺ oxidoreductase,pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxinoxidoreductase (OFOR). The H. thermophilus gene fdx1 encodes a[4Fe-4S]-type ferredoxin that is required for the reversiblecarboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR,respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). Theferredoxin associated with the Sulfolobus solfataricus2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S]type ferredoxin (Park et al. 2006). While the gene associated with thisprotein has not been fully sequenced, the N-terminal domain shares 93%homology with the zfx ferredoxin from S. acidocaldarius. The E. coligenome encodes a soluble ferredoxin of unknown physiological function,fdx. Some evidence indicates that this protein can function iniron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additionalferredoxin proteins have been characterized in Helicobacter pylori(Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et al.2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been clonedand expressed in E. coli (Fujinaga and Meyer, Biochemical andBiophysical Research Communications, 192(3): (1993)). Acetogenicbacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7,Clostridium ljungdahli and Rhodospirillum rubrum are predicted to encodeseveral ferredoxins, listed below.

Protein GenBank ID GI Number Organism fdx1 BAE02673.1 68163284Hydrogenobacter thermophilus M11214.1 AAA83524.1 144806 Clostridiumpasteurianum Zfx AAY79867.1 68566938 Sulfolobus acidocalarius FdxAAC75578.1 1788874 Escherichia coli hp_0277 AAD07340.1 2313367Helicobacter pylori fdxA CAL34484.1 112359698 Campylobacter jejuniMoth_0061 ABC18400.1 83571848 Moorella thermoacetica Moth_1200ABC19514.1 83572962 Moorella thermoacetica Moth_1888 ABC20188.1 83573636Moorella thermoacetica Moth_2112 ABC20404.1 83573852 Moorellathermoacetica Moth_1037 ABC19351.1 83572799 Moorella thermoaceticaCcarbDRAFT_4383 ZP_05394383.1 255527515 Clostridium carboxidivorans P7CcarbDRAFT_2958 ZP_05392958.1 255526034 Clostridium carboxidivorans P7CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridium carboxidivorans P7CcarbDRAFT_5296 ZP_05395295.1 255528511 Clostridium carboxidivorans P7CcarbDRAFT_1615 ZP_05391615.1 255524662 Clostridium carboxidivorans P7CcarbDRAFT_1304 ZP_05391304.1 255524347 Clostridium carboxidivorans P7cooF AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxNCAA35699.1 46143 Rhodobacter capsulatus Rru_A2264 ABC23064.1 83576513Rhodospirillum rubrum Rru_A1916 ABC22716.1 83576165 Rhodospirillumrubrum Rru_A2026 ABC22826.1 83576275 Rhodospirillum rubrum cooFAAC45122.1 1498747 Rhodospirillum rubrum fdxN AAA26460.1 152605Rhodospirillum rubrum Alvin_2884 ADC63789.1 288897953 Allochromatiumvinosum DSM 180 Fdx YP_002801146.1 226946073 Azotobacter vinelandii DJCKL_3790 YP_001397146.1 153956381 Clostridium kluyveri DSM 555 fer1NP_949965.1 39937689 Rhodopseudomonas palustris CGA009 Fdx CAA12251.13724172 Thauera aromatica CHY_2405 YP_361202.1 78044690 Carboxydothermushydrogenoformans Fer YP_359966.1 78045103 Carboxydothermushydrogenoformans Fer AAC83 945.1 1146198 Bacillus subtilis fdx1NP_249053.1 15595559 Pseudomonas aeruginosa PA01 yfhL AP_003148.189109368 Escherichia coli K-12 CLJU_c00930 ADK13195.1 300433428Clostridium ljungdahli CLJU_c00010 ADK13115.1 300433348 Clostridiumljungdahli CLJU_c01820 ADK13272.1 300433505 Clostridium ljungdahliCLJU_c17980 ADK14861.1 300435094 Clostridium ljungdahli CLJU_c17970ADK14860.1 300435093 Clostridium ljungdahli CLJU_c22510 ADK15311.1300435544 Clostridium ljungdahli CLJU_c26680 ADK15726.1 300435959Clostridium ljungdahli CLJU_c29400 ADK15988.1 300436221 Clostridiumljungdahli

Ferredoxin oxidoreductase enzymes transfer electrons from ferredoxins orflavodoxins to NAD(P)H. Two enzymes catalyzing the reversible transferof electrons from reduced ferredoxins to NAD(P)+ are ferredoxin:NAD+oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP+ oxidoreductase (FNR,EC 1.18.1.2). Ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) has anoncovalently bound FAD cofactor that facilitates the reversibletransfer of electrons from NADPH to low-potential acceptors such asferredoxins or flavodoxins (Blaschkowski et al., Eur. J Biochem.123:563-569 (1982); Fujii et al., 1977). The Helicobacter pylori FNR,encoded by HP1164 (fqrB), is coupled to the activity ofpyruvate:ferredoxin oxidoreductase (PFOR) resulting in thepyruvate-dependent production of NADPH (St et al. 2007). An analogousenzyme is found in Campylobacter jejuni (St Maurice et al., J.Bacteriol. 189:4764-4773 (2007)). A ferredoxin:NADP+ oxidoreductaseenzyme is encoded in the E. coli genome by fpr (Bianchi et al. 1993).Ferredoxin:NAD+ oxidoreductase utilizes reduced ferredoxin to generateNADH from NAD+. In several organisms, including E. coli, this enzyme isa component of multifunctional dioxygenase enzyme complexes. Theferredoxin:NAD+ oxidoreductase of E. coli, encoded by hcaD, is acomponent of the 3-phenylproppionate dioxygenase system involved ininvolved in aromatic acid utilization (Diaz et al. 1998).NADH:ferredoxin reductase activity was detected in cell extracts ofHydrogenobacter thermophilus, although a gene with this activity has notyet been indicated (Yoon et al. 2006). Additional ferredoxin:NAD(P)+oxidoreductases have been annotated in Clostridium carboxydivorans P7.The NADH-dependent reduced ferredoxin: NADP oxidoreductase of C.kluyveri, encoded by nfnAB, catalyzes the concomitant reduction offerredoxin and NAD+ with two equivalents of NADPH (Wang et al, JBacteriol 192: 5115-5123 (2010)). Finally, the energy-conservingmembrane-associated Rnf-type proteins (Seedorf et al, PNAS 105:2128-2133(2008); and Herrmann, J. Bacteriol 190:784-791 (2008)) provide a meansto generate NADH or NADPH from reduced ferredoxin.

Protein GenBank ID GI Number Organism fqrB NP_207955.1 15645778Helicobacter pylori fqrB YP_001482096.1 157414840 Campylobacter jejuniRPA3954 CAE29395.1 39650872 Rhodopseudomonas palustris Fpr BAH29712.1225320633 Hydrogenobacter thermophilus yumC NP_391091.2 255767736Bacillus subtilis Fpr P28861.4 399486 Escherichia coli hcaD AAC75595.11788892 Escherichia coli LOC100282643 NP_001149023.1 226497434 Zea maysNfnA YP_001393861.1 153953096 Clostridium kluyveri NfnB YP_001393862.1153953097 Clostridium kluyveri CcarbDRAFT_2639 ZP_05392639.1 255525707Clostridium carboxidivorans P7 CcarbDRAFT_2638 ZP_05392638.1 255525706Clostridium carboxidivorans P7 CcarbDRAFT_2636 ZP_05392636.1 255525704Clostridium carboxidivorans P7 CcarbDRAFT_5060 ZP_05395060.1 255528241Clostridium carboxidivorans P7 CcarbDRAFT_2450 ZP_05392450.1 255525514Clostridium carboxidivorans P7 CcarbDRAFT_1084 ZP_05391084.1 255524124Clostridium carboxidivorans P7 RnfC EDK33306.1 146346770 Clostridiumkluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnfG EDK33308.1146346772 Clostridium kluyveri RnfE EDK33309.1 146346773 Clostridiumkluyveri RnfA EDK33310.1 146346774 Clostridium kluyveri RnfB EDK33311.1146346775 Clostridium kluyveri CLJU_c11410 (RnfB) ADK14209.1 300434442Clostridium ljungdahlii CLJU_c11400 (RnfA) ADK14208.1 300434441Clostridium ljungdahlii CLJU_c11390 (RnfE) ADK14207.1 300434440Clostridium ljungdahlii CLJU_c11380 (RnfG) ADK14206.1 300434439Clostridium ljungdahlii CLJU_c11370 (RnfD) ADK14205.1 300434438Clostridium ljungdahlii CLJU_c11360 (RnfC) ADK14204.1 300434437Clostridium ljungdahlii MOTH_1518 (NfnA) YP_430370.1 83590361 Moorellathermoacetica MOTH_1517(NfnB) YP_430369.1 83590360 Moorellathermoacetica CHY_1992 (NfnA) YP_360811.1 78045020 Carboxydothermushydrogenoformans CHY_1993 (NfnB) YP_360812.1 78044266 Carboxydothermushydrogenoformans CLJU_c37220 (NfnAB) YP_003781850.1 300856866Clostridium ljungdahlii

FIG. 1, Step I—Formate Dehydrogenase (EM8)

Formate dehydrogenase (FDH; EM8) catalyzes the reversible transfer ofelectrons from formate to an acceptor. Enzymes with FDH activity utilizevarious electron carriers such as, for example, NADH (EC 1.2.1.2), NADPH(EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) and EM16s(EC 1.1.99.33). FDH enzymes have been characterized from Moorellathermoacetica (Andreesen and Ljungdahl, J Bacteriol 116:867-873 (1973);Li et al., J Bacteriol 92:405-412 (1966); Yamamoto et al., J Biol Chem.258:1826-1832 (1983). The loci, Moth_2312 is responsible for encodingthe alpha subunit of EM8 while the beta subunit is encoded by Moth_2314(Pierce et al., Environ Microbiol (2008)). Another set of genes encodingEM8 activity with a propensity for CO₂ reduction is encoded by Sfum_2703through Sfum_2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur JBiochem. 270:2476-2485 (2003)); Reda et al., PNAS 105:10654-10658(2008)). A similar set of genes presumed to carry out the same functionare encoded by CHY_0731, CHY_0732, and CHY_0733 in C. hydrogenoformans(Wu et al., PLoS Genet 1:e65 (2005)). EM8s are also found manyadditional organisms including C. carboxidivorans P7, Bacillusmethanolicus, Burkholderia stabilis, Moorella thermoacetica ATCC 39073,Candida boidinii, Candida methylica, and Saccharomyces cerevisiae S288c.The soluble EM8 from Ralstonia eutropha reduces NAD⁺ (fdsG, -B, -A, -C,-D) (Oh and Bowien, 1998).

Several EM8 enzymes have been identified that have higher specificityfor NADP as the cofactor as compared to NAD. This enzyme has been deemedas the NADP-dependent formate dehydrogenase and has been reported from 5species of the Burkholderia cepacia complex. It was tested and verifiedin multiple strains of Burkholderia multivorans, Burkholderia stabilis,Burkholderia pyrrocinia, and Burkholderia cenocepacia (Hatrongjit etal., Enzyme and Microbial Tech., 46: 557-561 (2010)). The enzyme fromBurkholderia stabilis has been characterized and the apparent K_(m) ofthe enzyme were reported to be 55.5 mM, 0.16 mM and 1.43 mM for formate,NADP, and NAD respectively. More gene candidates can be identified usingsequence homology of proteins deposited in Public databases such asNCBI, MI and the metagenomic databases.

Protein GenBank ID GI Number Organism Moth_2312 YP_431142 148283121Moorella thermoacetica Moth_2314 YP_431144 83591135 Moorellathermoacetica Sfum_2703 YP_846816.1 116750129 Syntrophobacterfumaroxidans Sfum_2704 YP_846817.1 116750130 Syntrophobacterfumaroxidans Sfum_2705 YP_846818.1 116750131 Syntrophobacterfumaroxidans Sfum_2706 YP_846819.1 116750132 Syntrophobacterfumaroxidans CHY_0731 YP_359585.1 78044572 Carboxydothermushydrogenoformans CHY_0732 YP_359586.1 78044500 Carboxydothermushydrogenoformans CHY_0733 YP_359587.1 78044647 Carboxydothermushydrogenoformans CcarbDRAFT_0901 ZP_05390901.1 255523938 Clostridiumcarboxidivorans P7 CcarbDRAFT_4380 ZP_05394380.1 255527512 Clostridiumcarboxidivorans P7 fdhA, EIJ82879.1 387590560 Bacillus methanolicus MGA3MGA3_06625 fdhA, PB1_11719 ZP_10131761.1 387929084 Bacillus methanolicusPB1 fdhD, MGA3_06630 EIJ82880.1 387590561 Bacillus methanolicus MGA3fdhD, PB1_11724 ZP_10131762.1 387929085 Bacillus methanolicus PB1 fdhACF35003.1 194220249 Burkholderia stabilis fdh ACF35004.1 194220251Burkholderia pyrrocinia fdh ACF35002.1 194220247 Burkholderiacenocepacia fdh ACF35001.1 194220245 Burkholderia multivorans fdhACF35000.1 194220243 Burkholderia cepacia FDH1 AAC49766.1 2276465Candida boidinii fdh CAA57036.1 1181204 Candida methylica FDH2 P0CF35.1294956522 Saccharomyces cerevisiae S288c FDH1 NP_015033.1 6324964Saccharomyces cerevisiae S288c fdsG YP 725156.1 113866667 Ralstoniaeutropha fdsB YP_725157.1 113866668 Ralstonia eutropha fdsA YP_725158.1113866669 Ralstonia eutropha fdsC YP_725159.1 113866670 Ralstoniaeutropha fdsD YP_725160.1 113866671 Ralstonia eutropha

FIG. 1, Step J—Methanol Dehydrogenase (EM9)

NAD+ dependent EM9 enzymes (EC 1.1.1.244) catalyze the conversion ofmethanol and NAD+ to formaldehyde and NADH. An enzyme with this activitywas first characterized in Bacillus methanolicus (Heggeset, et al.,Applied and Environmental Microbiology, 78(15):5170-5181 (2012)). Thisenzyme is zinc and magnesium dependent, and activity of the enzyme isenhanced by the activating enzyme encoded by act (Kloosterman et al, JBlot Chem 277:34785-92 (2002)). The act is a Nudix hydrolase. Several ofthese candidates have been identified and shown to have activity onmethanol. Additional NAD(P)+ dependent enzymes can be identified bysequence homology. EM9 enzymes utilizing different electron acceptorsare also known in the art. Examples include cytochrome dependent enzymessuch as mxalF of the methylotroph Methylobacterium extorquens (Nunn etal, Nucl Acid Res 16:7722 (1988)). EM9 enzymes of methanotrophs such asMethylococcus capsulatis function in a complex with methanemonooxygenase (MMO) (Myronova et al., Biochem 45:11905-14 (2006)).Methanol can also be oxidized to formaldehyde by alcohol oxidase enzymessuch as methanol oxidase (EC 1.1.3.13) of Candida boidinii (Sakai etal., Gene 114: 67-73 (1992)).

Protein GenBank ID GI Number Organism mdh, MGA3_17392 EIJ77596.1387585261 Bacillus methanolicus MGA3 mdh2, MGA3_07340 EIJ83020.1387590701 Bacillus methanolicus MGA3 mdh3, MGA3_10725 EIJ80770.1387588449 Bacillus methanolicus MGA3 act, MGA3_09170 EIJ83380.1387591061 Bacillus methanolicus MGA3 mdh, PB1_17533 ZP_10132907.1387930234 Bacillus methanolicus PB1 mdh1, PB1_14569 ZP_10132325.1387929648 Bacillus methanolicus PB1 mdh2, PB1_12584 ZP_10131932.1387929255 Bacillus methanolicus PB1 act, PB1_14394 ZP_10132290.1387929613 Bacillus methanolicus PB1 BFZC1_05383 ZP_07048751.1 299535429Lysinibacillus fusiformis BFZC1_20163 ZP_07051637.1 299538354Lysinibacillus fusiformis Bsph_4187 YP_001699778.1 169829620Lysinibacillus sphaericus Bsph_1706 YP_001697432.1 169827274Lysinibacillus sphaericus mdh2 YP_004681552.1 339322658 Cupriavidusnecator N-1 nudF1 YP_004684845.1 339325152 Cupriavidus necator N-1BthaA_010200007655 ZP_05587334.1 257139072 Burkholderia thailandensisE264 BTH_11076 YP_441629.1 83721454 Burkholderia thailandensis(MutT/NUDIX NTP E264 pyrophosphatase) BalcAV_11743 ZP_10819291.1402299711 Bacillus alcalophilus ATCC 27647 BalcAV_05251 ZP_10818002.1402298299 Bacillus alcalophilus ATCC 27647 alcohol dehydrogenaseYP_725376.1 113866887 Ralstonia eutropha H16 alcohol dehydrogenaseYP_001447544 156976638 Vibrio harveyi ATCC BAA-1116 P3TCK_27679ZP_01220157.1 90412151 Photobacterium profundum 3TCK alcoholdehydrogenase YP_694908 110799824 Clostridium perfringens ATCC 13124adhB NP_717107 24373064 Shewanella oneidensis MR-1 alcohol dehydrogenaseYP_237055 66047214 Pseudomonas syringae pv. syringae B728a alcoholdehydrogenase YP_359772 78043360 Carboxydothermus hydrogenoformansZ-2901 alcohol dehydrogenase YP_003990729 312112413 Geobacillus sp.Y4.1MC1 PpeoK3_010100018471 ZP_10241531.1 390456003 Paenibacilluspeoriae KCTC 3763 OBE_12016 EKC54576 406526935 human gut metagenomealcohol dehydrogenase YP_003310546 269122369 Sebaldella termitidis ATCC33386 alcohol dehydrogenase YP_001343716 152978087 Actinobacillussuccinogenes 130Z dhaT AAC45651 2393887 Clostridium pasteurianum DSM 525alcohol dehydrogenase NP_561852 18309918 Clostridium perfringens str. 13BAZO_10081 ZP_11313277.1 410459529 Bacillus azotoformans LMG 9581alcohol dehydrogenase YP_007491369 452211255 Methanosarcina mazei Tuc01alcohol dehydrogenase YP_004860127 347752562 Bacillus coagulans 36D1alcohol dehydrogenase YP_002138168 197117741 Geobacter bemidjiensis BemDesmeDRAFT_1354 ZP_08977641.1 354558386 Desulfitobacteriummetallireducens DSM 15288 alcohol dehydrogenase YP_001337153 152972007Klebsiella pneumoniae subsp. pneumoniae MGH 78578 alcohol dehydrogenaseYP_001113612 134300116 Desulfotomaculum reducens Ml-1 alcoholdehydrogenase YP_001663549 167040564 Thermoanaerobacter sp. X514ACINNAV82_2382 ZP_16224338.1 421788018 Acinetobacter baumannii Naval-82DVU2405 YP_011618 46580810 Desulfovibrio vulgaris str. Hildenboroughalcohol dehydrogenase YP_005052855 374301216 Desulfovibrio africanusstr. Walvis Bay alcohol dehydrogenase YP_002434746 218885425Desulfovibrio vulgaris str. ‘Miyazaki F’ alcohol dehydrogenase AGF87161451936849 uncultured organism DesfrDRAFT_3929 ZP_07335453.1 303249216Desulfovibrio fructosovorans JJ alcohol dehydrogenase NP_617528 20091453Methanosarcina acetivorans C2A alcohol dehydrogenase NP_343875.115899270 Sulfolobus solfataricus P-2 adh4 YP_006863258 408405275Nitrososphaera gargensis Ga9.2 BD31_10957 ZP_10117398.1 386875211Nitrosopumilus salaria BD31 alcohol dehydrogenase YP_004108045.1316933063 Rhodopseudomonas palustris DX-1 Ta0841 NP_394301.1 16081897Thermoplasma acidophilum PTO1151 YP_023929.1 48478223 Picrophilustorridus DSM9790 alcohol dehydrogenase ZP_10129817.1 387927138 Bacillusmethanolicus PB-1 cgR_2695 YP_001139613.1 145296792 Corynebacteriumglutamicum R alcohol dehydrogenase YP_004758576.1 340793113Corynebacterium variabile HMPREF1015_01790 ZP_09352758.1 365156443Bacillus smithii ADH1 NP_014555.1 6324486 Saccharomyces cerevisiaeNADH-dependent YP_001126968.1 138896515 Geobacillus butanoldehydrogenase themodenitrificans NG80-2 A alcohol dehydrogenaseWP_007139094.1 494231392 Flavobacterium frigoris methanol WP_003897664.1 489994607 Mycobacterium smegmatis dehydrogenase ADH1BNP_000659.2 34577061 Homo sapiens PMI01_01199 ZP_10750164.1 399072070Caulobacter sp. AP07 BurJ1DRAFT_3901 ZP_09753449.1 375107188Burkholderiales bacterium Joshi_001 YiaY YP_026233.1 49176377Escherichia coli MCA0299 YP_112833.1 53802410 Methylococcus capsulatisMCA0782 YP_113284.1 53804880 Methylococcus capsulatis mxaIYP_002965443.1 240140963 Methylobacterium extorquens mxaF YP_002965446.1240140966 Methylobacterium extorquens AOD1 AAA34321.1 170820 Candidaboidinii

An in vivo assay was developed to determine the activity of methanoldehydrogenases. This assay relies on the detection of formaldehyde(HCHO), thus measuring the forward activity of the enzyme (oxidation ofmethanol). To this end, a strain comprising a BDOP and lacking frmA,frmB, frmR was created using Lamba Red recombinase technology (Datsenkoand Wanner, Proc. Natl. Acad. Sci. USA, 6 97(12): 6640-5 (2000).Plasmids expressing methanol dehydrogenases were transformed into thestrain, then grown to saturation in LB medium+ antibiotic at 37° C. withshaking. Transformation of the strain with an empty vector served as anegative control. Cultures were adjusted by O.D. and then diluted 1:10into M9 medium+0.5% glucose+ antibiotic and cultured at 37° C. withshaking for 6-8 hours until late log phase. Methanol was added to 2% v/vand the cultures were further incubated for 30 min. with shaking at 37°C. Cultures were spun down and the supernatant was assayed forformaldehyde produced using DETECTX Formaldehyde Detection kit (ArborAssays; Ann Arbor, Mich.) according to manufacturer's instructions. ThefrmA, frmB, frmR deletions resulted in the native formaldehydeutilization pathway to be deleted, which enables the formation offormaldehyde that can be used to detect methanol dehydrogenase activityin the NNOMO.

The activity of several enzymes was measured using the assay describedabove. The results of four independent experiments are provided in Table1 below.

TABLE 1 Results of in vivo assays showing formaldehyde (HCHO) productionby various NNOMO comprising a plasmid expressing a methanoldehydrogenase. Accession HCHO Accession HCHO Accession HCHO AccessionHCHO number (μm) number (μM) number (μM) number (μM) Experiment 1Experiment 2 Experiment 3 Experiment 4 EIJ77596.1 >50 EIJ77596.1 >50EIJ77596.1 >50 EIJ77596.1 >50 EIJ83020.1 >20 NP_00659.2 >50NP_561852 >50 ZP_10241531.1 >50 EIJ80770.1 >50 YP_004758576.1 >20YP_002138168 >50 YP_005052855 >50 ZP_10132907.1 >20 ZP_09352758.1 >50YP_026233.1 >50 ZP_10132907.1 >50 ZP_10132325.1 >20 ZP_10129817.1 >20YP_001447544 >50 NP_617528 >50 ZP_10131932.1 >50 YP_001139613.1 >20Metalibrary >50 NP_617528 >50 ZP_07048751.1 >50 NP_014555.1 >10YP_359772 >50 ZP_08977641.1 >20 YP_001699778.1 >50 WP_007139094.1 >10ZP_01220157.1 >50 YP_237055 >20 YP_004681552.1 >10 NP_343875.1 >1ZP_07335453.1 >20 Empty vector 49.36 ZP_10819291.1 <1 YP_006863258 >1YP_001337153 >20 MT vector 2.33 NP_394301.1 >1 YP_694908 >20ZP_10750164.1 >1 NP_717107 >20 YP_023929.1 >1 AAC45651 >10 ZP_08977641.1<1 ZP_11313277.1 >10 ZP_10117398.1 <1 ZP_16224338.1 >10 YP_004108045.1<1 YP_001113612 >10 ZP_09753449.1 <1 YP_004860127 >10 MT vector 0.17YP_003310546 >10 YP_001343716 >10 NP_717107 >10 YP_002434746 >10 Emptyvector 0.11

FIG. 1, Step K—Spontaneous or Formaldehyde Activating Enzyme (EM10)

The conversion of formaldehyde and THF to methylenetetrahydrofolate canoccur spontaneously. It is also possible that the rate of this reactioncan be enhanced by an EM10. A formaldehyde activating enzyme (Fae; EM10)has been identified in Methylobacterium extorquens AM1 which catalyzesthe condensation of formaldehyde and tetrahydromethanopterin tomethylene tetrahydromethanopterin (Vorholt, et al., J. Bacteriol.,182(23), 6645-6650 (2000)). It is possible that a similar enzyme existsor can be engineered to catalyze the condensation of formaldehyde andtetrahydrofolate to methylenetetrahydrofolate. Homologs exist in severalorganisms including Xanthobacter autotrophicus Py2 and Hyphomicrobiumdenitrificans ATCC 51888.

Protein GenBank ID GI Number Organism MexAM1_META1p1766 Q9FA38.317366061 Methylobacterium extorquens AM1 Xaut_0032 YP_001414948.1154243990 Xanthobacter autotrophicus Py2 Hden_1474 YP_003755607.1300022996 Hyphomicrobium denitrificans ATCC 51888

FIG. 1, Step L—Formaldehyde Dehydrogenase (EM11)

Oxidation of formaldehyde to formate is catalyzed by EM11. A NAD+dependent EM11 enzyme is encoded by fdhA of Pseudomonas putida (Ito etal, J Bacteriol 176: 2483-2491 (1994)). Additional EM11 enzymes includethe NAD+ and glutathione independent EM11 from Hyphomicrobium zavarzinii(Jerome et al, Appl Microbiol Biotechnol 77:779-88 (2007)), theglutathione dependent EM11 of Pichia pastoris (Sunga et al, Gene330:39-47 (2004)) and the NAD(P)+ dependent EM11 of Methylobactermarinus (Speer et al, FEMS Microbiol Lett, 121(3):349-55 (1994)).

Protein GenBank ID GI Number Organism fdhA P46154.3 1169603 Pseudomonasputida faoA CAC85637.1 19912992 Hyphomicrobium zavarzinii Fld1CCA39112.1 328352714 Pichia pastoris fdh P47734.2 221222447Methylobacter marinus

In addition to the EM11 enzymes listed above, alternate enzymes andpathways for converting formaldehyde to formate are known in the art.For example, many organisms employ glutathione-dependent formaldehydeoxidation pathways, in which formaldehyde is converted to formate inthree steps via the intermediates S-hydroxymethylglutathione andS-formylglutathione (Vorholt et al, J Bacteriol 182:6645-50 (2000)). Theenzymes of this pathway are EM12 (EC 4.4.1.22), EM13 (EC 1.1.1.284) andEM14 (EC 3.1.2.12).

FIG. 1, Step M—Spontaneous or S-(Hydroxymethyl)Glutathione Synthase(EM12)

While conversion of formaldehyde to S-hydroxymethylglutathione can occurspontaneously in the presence of glutathione, it has been shown byGoenrich et al (Goenrich, et al., J Biol Chem 277(5); 3069-72 (2002))that an enzyme from Paracoccus denitrificans can accelerate thisspontaneous condensation reaction. The enzyme catalyzing the conversionof formaldehyde and glutathione was purified and namedglutathione-dependent formaldehyde-activating enzyme (Gfa). The geneencoding it, which was named gfa, is located directly upstream of thegene for EM13, which catalyzes the subsequent oxidation ofS-hydroxymethylglutathione. Putative proteins with sequence identity toGfa from P. denitrificans are present also in Rhodobacter sphaeroides,Sinorhizobium meliloti, and Mesorhizobium loti.

Protein GenBank ID GI Number Organism Gfa Q51669.3 38257308 Paracoccusdenitrificans Gfa ABP71667.1 145557054 Rhodobacter sphaeroides ATCC17025 Gfa Q92WX6.1 38257348 Sinorhizobium meliloti 1021 Gfa Q98LU4.238257349 Mesorhizobium loti MAFF303099

FIG. 1, Step N—Glutathione-Dependent Formaldehyde Dehydrogenase (EM13)

EM13 (GS-FDH) belongs to the family of class III alcohol dehydrogenases.Glutathione and formaldehyde combine non-enzymatically to formhydroxymethylglutathione, the true substrate of the GS-FDH catalyzedreaction. The product, S-formylglutathione, is further metabolized toformic acid.

Protein GenBankID GI Number Organism frmA YP_488650.1 388476464Escherichia coli K-12 MG1655 SFA1 NP_010113.1 6320033 Saccharomycescerevisiae S288c flhA AAC44551.1 1002865 Paracoccus denitrificans adhIAAB09774.1 986949 Rhodobacter sphaeroides

FIG. 1, Step O—S-Formylglutathione Hydrolase (EM14)

EM14 is a glutathione thiol esterase found in bacteria, plants andanimals. It catalyzes conversion of S-formylglutathione to formate andglutathione. The fghA gene of P. denitrificans is located in the sameoperon with gfa and flhA, two genes involved in the oxidation offormaldehyde to formate in this organism. In E. coli, FrmB is encoded inan operon with FrmR and FrmA, which are proteins involved in theoxidation of formaldehyde. YeiG of E. coli is a promiscuous serinehydrolase; its highest specific activity is with the substrateS-formylglutathione.

Protein GenBank ID GI Number Organism frmB NP_414889.1 16128340Escherichia coli K-12 MG1655 yeiG AAC75215.1 1788477 Escherichia coliK-12 MG1655 fghA AAC44554.1 1002868 Paracoccus denitrificans

4.2 Example II—Enhanced Yield of 1,4 Butanediol from Carbohydrates usingMethanol

Exemplary MMPs for enhancing the availability of reducing equivalentsare provided in FIG. 2.

FIG. 2, Step A—Succinyl-CoA Transferase (EB1) or Succinyl-CoA Synthetase(EB2A) (or Succinyl-CoA Ligase)

The conversion of succinate to succinyl-CoA is catalyzed by EB1 or EB2A(ligase). EB1 enzymes include cat1 of Clostridium kluyveri and ygfH ofE. coli (Seedorf et al., Proc. Natl. Acad. Sci U.S.A 105:2128-2133(2008); Sohling et al., J Bacteriol. 178:871-880 (1996); Haller et al.,Biochemistry, 39(16) 4622-4629). Homologs can be found in, for example,Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonaeserovar, and Yersinia intermedia ATCC 29909. Succinyl-CoA:3:oxoacid-CoAtransferase employs succinate as the CoA acceptor. This enzyme isencoded by pcaI and pcaJ in Pseudomonas putida (Kaschabek et al., JBacteriol. 184:207-215 (2002)). Similar enzymes are found inAcinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)),Streptomyces coelicolor and Pseudomonas knackmussii (formerly sp. B13)(Gobel et al., J Bacteriol. 184:216-223 (2002); Kaschabek et al., JBacteriol. 184:207-215 (2002)). Other succinyl-CoA:3:oxoacid-CoAtransferases have been characterized in Helicobacter pylori(Corthesy-Theulaz et al., J Biol. Chem. 272:25659-25667 (1997)),Bacillus subtilis (Stols et al., Protein Expr. Purif. 53:396-403 (2007))and Homo sapiens (Fukao, T., et al., Genomics 68:144-151 (2000); Tanaka,H., et al., Mol Hum Reprod 8:16-23 (2002)). GenBank information relatedto these genes is summarized below.

Protein GenBank ID GI Number Organism cat1 P38946.1 729048 Clostridiumkluyveri ygfH NP_417395.1 16130821 Escherichia coli CIT292_04485ZP_03838384.1 227334728 Citrobacter youngae SARI_04582 YP_001573497.1161506385 Salmonella enterica yinte0001_14430 ZP_04635364.1 238791727Yersinia intermedia peaI 24985644 AAN69545.1 Pseudomonas putida pcaJ26990657 NP_746082.1 Pseudomonas putida peaI 50084858 YP_046368.1Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1 Acinetobacter sp. ADP1peaI 21224997 NP_630776.1 Streptomyces coelicolor pcaJ 21224996NP_630775.1 Streptomyces coelicolor catI 75404583 Q8VPF3 Pseudomonasknackmussii catJ 75404582 Q8VPF2 Pseudomonas knackmussii HPAG1_0676108563101 YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418Aelicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB16080949 NP_391777 Bacillus subtilis OXCT1 NP_000427 4557817 Homosapiens OXCT2 NP_071403 11545841 Homo sapiens

EB2A, also called succinyl-CoA ligase, is encoded by sucCD of E. coliand LSC1 and LSC2 genes of Saccharomyces cerevisiae. These enzymescatalyze the formation of succinyl-CoA from succinate with theconcomitant consumption of one ATP in a reaction which is reversible invivo (Buck et al., Biochemistry 24:6245-6252 (1985)).

Protein GenBank ID GI Number Organism sucC NP_415256.1 16128703Escherichia coli sucD AAC73823.1 1786949 Escherichia coli LSC1 NP_0147856324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomycescerevisiae

FIG. 2, Step B—Succinyl-CoA Reductase (Aldehyde Forming) (EB3)

Enzymes with succinyl-CoA reductase activity are encoded by sucD ofClostridium kluyveri (Sohling, J. Bacteriol. 178:871-880 (1996)) andsucD of Porphyromonas gingivalis (Takahashi, J. Bacteriol 182:4704-4710(2000)). Additional succinyl-CoA reductase enzymes participate in the3-hydroxypropionate/4-HB cycle of thermophilic archaea such asMetallosphaera sedula (Berg et al., Science 318:1782-1786 (2007)) andThermoproteus neutrophilus (Ramos-Vera et al., J Bacteriol,191:4286-4297 (2009)). The M. sedula enzyme, encoded by Msed_0709, isstrictly NADPH-dependent and also has malonyl-CoA reductase activity.The T. neutrophilus enzyme is active with both NADPH and NADH.

Protein GenBank ID GI Number Organism MSED_0709 YP_001190808.1 146303492Metallosphaera sedula Tneu_0421 ACB39369.1 170934108 Thermoproteusneutrophilus sucD P38947.1 172046062 Clostridium kluyveri sucDNP_904963.1 34540484 Porphyromonas gingivalis

FIG. 2, Step C—4-Hydroxybutyrate Dehydrogenase (EB4)

Enzymes exhibiting EB4 activity (EC 1.1.1.61) have been characterized inRalstonia eutropha (Bravo et al., J. Forensic Sci. 49:379-387 (2004),Clostridium kluyveri (Wolff and Kenealy, Protein Expr. Purif. 6:206-212(1995)) and Arabidopsis thaliana (Breitkreuz et al., J. Biol. Chem.278:41552-41556 (2003)). Other EB4 enzymes are found in Porphyromonasgingivalis and gbd of an uncultured bacterium. Accession numbers ofthese genes are listed in the table below.

Protein GenBank ID GI Number Organism 4hbd YP_726053.1 113867564Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM555 4hbd Q94B07 75249805 Arabidopsis thaliana 4-hBd NP_904964.1 34540485Porphyromonas gingivalis W83 gbd AF148264.1 5916168 Uncultured bacterium

FIG. 2, Step D—Hydroxybutyrate Kinase

Activation of 4-HB to 4-hydroxybutyryl-phosphate is catalyzed by EB5.Phosphotransferase enzymes in the EC class 2.7.2 transform carboxylicacids to phosphonic acids with concurrent hydrolysis of one ATP. Enzymessuitable for catalyzing this reaction include butyrate kinase, acetatekinase, aspartokinase and gamma-glutamyl kinase. Butyrate kinase carriesout the reversible conversion of butyryl-phosphate to butyrate duringacidogenesis in C. acetobutylicum (Cary et al., Appl. Environ.Microbiol. 56:1576-1583 (1990)). This enzyme is encoded by either of thetwo buk gene products (Huang et al., J. Mol. Microbiol. Biotechnol.2:33-38 (2000)). Other butyrate kinase enzymes are found in C.butyricum, C. beijerinckii and C. tetanomorphum (Twarog and Wolfe, J.Bacteriol. 86:112-117 (1963)). A related enzyme, isobutyrate kinase fromThermotoga maritime, has also been expressed in E. coli and crystallized(Diao et al., Acta Crystallo. D. Biol. Crystallo. 59:1100-1102 (2003);Diao and Hasson, J. Bacteriol. 191:2521-2529 (2009)). Aspartokinasecatalyzes the ATP-dependent phosphorylation of aspartate andparticipates in the synthesis of several amino acids. The aspartokinaseIII enzyme in E. coli, encoded by lysC, has a broad substrate range, andthe catalytic residues involved in substrate specificity have beenelucidated (Keng and Viola, Arch. Biochem. Biophys. 335:73-81 (1996)).Two additional kinases in E. coli are also good candidates: acetatekinase and gamma-glutamyl kinase. The E. coli acetate kinase, encoded byackA (Skarstedt and Silverstein, J. Biol. Chem. 251:6775-6783 (1976)),phosphorylates propionate in addition to acetate (Hesslinger et al.,Mol. Microbiol. 27:477-492 (1998)). The E. coli gamma-glutamyl kinase,encoded by proB (Smith et al., J. Bacteriol. 157:545-551 (1984)),phosphorylates the gamma carbonic acid group of glutamate.

Gene Accession No. GI No. Organism buk1 NP_349675 15896326 Clostridiumacetobutylicum buk2 Q97II1 20137415 Clostridium acetobutylicum buk2Q9X278.1 6685256 Thermotoga maritima lysC NP_418448.1 16131850Escherichia coli ackA NP_416799.1 16130231 Escherichia coli proBNP_414777.1 16128228 Escherichia coli buk YP_001307350.1 150015096Clostridium beijerhickii buk2 YP_001311072.1 150018818 Clostridiumbeijerhickii

FIG. 2, Step E—Phosphotrans-4-Hydroxybutyrylase (EB6)

EB6 catalyzes the transfer of the 4-hydroxybutyryl group from phosphateto CoA. Acyltransferases suitable for catalyzing this reaction includephosphotransacetylase and phosphotransbutyrylase. The pta gene from E.coli encodes an enzyme that can convert acetyl-phosphate into acetyl-CoA(Suzuki, Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme canalso utilize propionyl-CoA instead of acetyl-CoA (Hesslinger et al.,Mol. Microbiol. 27:477-492 (1998)). Similarly, the ptb gene from C.acetobutylicum encodes an enzyme that can convert butyryl-CoA intobutyryl-phosphate (Walter et al., Gene 134:107-111 (1993)); Huang etal., J Mol. Microbiol. Biotechnol. 2:33-38 (2000). Additional ptb genescan be found in Clostridial organisms, butyrate-producing bacteriumL2-50 (Louis et al., J. Bacteriol. 186:2099-2106 (2004)) and Bacillusmegaterium (Vazquez et al., Curr. Microbiol 42:345-349 (2001)).

Gene Accession No. GI No. Organism pta NP_416800.1 16130232 Escherichiacoli ptb NP_349676 15896327 Clostridium acetobutylicum ptbYP_001307349.1 150015095 Clostridium beijerinckii ptb AAR19757.138425288 butyrate-producing bacterium L2-50 ptb CAC07932.1 10046659Bacillus megaterium

FIG. 2, Step F—4-Hydroxybutyryl-CoA Reductase (Aldehyde Forming) (EB7)

4-hydroxybutyryl-CoA reductase catalyzes the reduction of4-hydroxybutyryl-CoA to its corresponding aldehyde. Several acyl-CoAdehydrogenases are capable of catalyzing this activity. The succinatesemialdehyde dehydrogenases (SucD) of Clostridium kluyveri and P.gingivalis were shown in ref (WO/2008/115840) to convert4-hydroxybutyryl-CoA to 4-hydroxybutanal as part of a pathway to produce1,4-butanediol. Many butyraldehyde dehydrogenases are also active on4-hydroxybutyraldehyde, including bld of Clostridiumsaccharoperbutylacetonicum and bphG of Pseudomonas sp (Powlowski et al.,J. Bacteriol. 175:377-385 (1993)). Yet another candidate is the ald genefrom Clostridium beijerinckii (Toth, Appl. Environ. Microbiol.65:4973-4980 (1999). This gene is very similar to eutE that encodesacetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth,Appl. Environ. Microbiol. 65:4973-4980 (1999). These and additionalproteins with 4-hydroxybutyryl-CoA reductase activity are identifiedbelow.

Protein GenBank ID GI Number Organism sucD P38947.1 172046062Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonas gingivalisbld AAP42563.1 31075383 Clostridium saccharoperbutylacetonicum bphGBAA03892.1 425213 Pseudomonas sp Ald AAT66436 49473535 Clostridiumbeijerinckii eutE AAA80209 687645 Salmonella typhimurium eutE P774452498347 Escherichia coli ald YP_001310903.1 150018649 Clostridiumbeijerinckii NCIMB 8052 Ald ZP_03778292.1 225569267 Clostridiumhylemonae DSM 15053 Ald ZP_03705305.1 225016072 Clostridiummethylpentosum DSM 5476 Ald ZP_03 715465.1 225026273 Eubacterium halliiDSM 3353 Ald ZP_01962381.1 153809713 Ruminococcus obeum ATCC 29174 AldYP_003701164.1 297585384 Bacillus selenitireducens MLS10 Ald AAP42563.131075383 Clostridium saccharoperbutylacetonicum N1-4 Ald YP_795711.1116334184 Lactobacillus brevis ATCC 367 Ald YP_002434126.1 218782808Desulfatibacillum alkenivorans AK-01 Ald YP_001558295.1 160879327Clostridium phytofermentans ISDg Ald ZP_02089671.1 160942363 Clostridiumbolteae ATCC BAA-613 Ald ZP_01222600.1 90414628 Photobacterium profundum3TCK Ald YP_001452373.1 157145054 Citrobacter koseri ATCC BAA-895 AldNP_460996.1 16765381 Salmonella enterica typhimurium Ald YP_003307836.1269119659 Sebaldella termitidis ATCC 33386 Ald ZP_04969437.1 254302079Fusobacterium nucleatum subsp. polymorphum ATCC 10953 Ald YP_002892893.1237808453 Tolumonas auensis DSM 9187 Ald YP_426002.1 83592250Rhodospirillum rubrum ATCC 11170FIG. 2, Step G—1,4-butanediol dehydrogenase (EB8)

EB8 catalyzes the reduction of 4-hydroxybutyraldehyde to 1,4-butanediol.Exemplary genes encoding this activity include alrA of Acinetobacter sp.strain M-1 (Tani et al., Appl. Environ. Microbiol. 66:5231-5235 (2000)),yqhD and fucO from E. coli (Sulzenbacher et al., J Mol Biol 342:489-502(2004)) and bdh I and bdh II from C. acetobutylicum (Walter et al, J.Bacteriol 174:7149-7158 (1992)). Additional EB8 enzymes are encoded bybdh in C. saccharoperbutylacetonicum and Cbei_1722, Cbei_2181 andCbei_2421 in C. beijerinckii. These and other enzymes with1,4-butanediol activity are listed in the table below.

Protein GenBank ID GI Number Organism alrA BAB12273.1 9967138Acinetobacter sp. strain M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae fucO NP_417279.1 16130706 Escherichia coli yqhD NP_417484.116130909 Escherichia coli bdh I NP_349892.1 15896543 Clostridiumacetobutylicum bdh II NP_349891.1 15896542 Clostridium acetobutylicumbdh BAF45463.1 124221917 Clostridium saccharoperbutylacetonicumCbei_1722 YP_001308850 150016596 Clostridium beijerinckii Cbei_2181YP_001309304 150017050 Clostridium beijerinckii Cbei_2421 YP_001309535150017281 Clostridium beijerinckii 14bdh AAC76047.1 1789386 Escherichiacoli K-12 MG1655 14bdh YP_001309304.1 150017050 Clostridium beijerinckiiNCIMB 8052 14bdh P13604.1 113352 Clostridium saccharobutylicum 14bdhZP_03760651.1 225405462 Clostridium asparagiforme DSM 15981 14bdhZP_02083621.1 160936248 Clostridium bolteae ATCC BAA-613 14bdhYP_003845251.1 302876618 Clostridium cellulovorans 743B 14bdhZP_03294286.1 210624270 Clostridium hiranonis DSM 13275 14bdhZP_03705769.1 225016577 Clostridium methylpentosum DSM 5476 14bdhYP_003179160.1 257783943 Atopobium parvulum DSM 20469 14bdhYP_002893476.1 237809036 Tolumonas auensis DSM 9187 14bdh ZP_05394983.1255528157 Clostridium carboxidivorans P7

FIG. 2, Step H—Succinate Reductase (EB9)

Direct reduction of succinate to succinate semialdehyde is catalyzed bya carboxylic acid reductase. Exemplary enzymes for catalyzing thistransformation are described below (see FIG. 2, Step K).

FIG. 2, Step I—Succinyl-CoA Reductase (Alcohol Forming) (EB10)

EB10 enzymes are bifunctional oxidoreductases that convert succinyl-CoAto 4-HB. EB15 enzymes candidates, described below (FIG. 2, Step M), arealso suitable for catalyzing the reduction of succinyl-CoA.

FIG. 2, Step J—4-Hydroxybutyryl-CoA Transferase (EB11) or4-Hydroxybutyryl-CoA Synthetase (EB12)

Conversion of 4-HB to 4-hydroxybutyryl-CoA is catalyzed by a CoAtransferase or synthetase. EB11 enzymes include the gene products ofcat1, cat2, and cat3 of Clostridium kluyveri (Seedorf et al., Proc.Natl. Acad. Sci U.S.A 105:2128-2133 (2008); Sohling et at., J Bacteriol.178:871-880 (1996)). Similar CoA transferase activities are also presentin Trichomonas vaginalis, Trypanosoma brucei, Clostridium aminobutyricumand Porphyromonas gingivalis (Riviere et al., J. Biol. Chem.279:45337-45346 (2004); van Grinsven et al., J. Biol. Chem.283:1411-1418 (2008)).

Protein GenBank ID GI Number Organism cat1 P38946.1 729048 Clostridiumkluyveri cat2 P38942.2 172046066 Clostridium kluyveri cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei cat2 CAB60036.1 6249316 Clostridium aminobutyricum cat2NP_906037.1 34541558 Porphyromonas gingivalis W83

4HB-CoA synthetase catalyzes the ATP-dependent conversion of 4-HB to4-hydroxybutyryl-CoA. AMP-forming 4-HB-CoA synthetase enzymes are foundin organisms that assimilate carbon via thedicarboxylate/hydroxybutyrate cycle or the 3-hydroxypropionate/4-HBcycle. Enzymes with this activity have been characterized inThermoproteus neutrophilus and Metallosphaera sedula (Ramos-Vera et al,J Bacteriol 192:5329-40 (2010); Berg et al, Science 318:1782-6 (2007)).Others can be inferred by sequence homology. ADP forming CoAsynthetases, such EB2A, are also suitable candidates.

Protein GenBank ID GI Number Organism Tneu_0420 ACB39368.1 170934107Thermoproteus neutrophilus Caur_0002 YP_001633649.1 163845605Chloroflexus aurantiacus J-10-fl Cagg_3790 YP_002465062 219850629Chloroflexus aggregans DSM 9485 acs YP_003431745 288817398Hydrogenobacter thermophilus TK-6 Pisl_0250 YP_929773.1 119871766Pyrobaculum islandicum DSM 4184 Msed_1422 ABP95580.1 145702438Metallosphaera sedula

FIG. 2, Step K—4-Hydroxybutyrate Reductase (EB13)

Reduction of 4-HB to 4-hydroxybutanal is catalyzed by a carboxylic acidreductase (CAR). Such an enzyme is found in Nocardia iowensis.Carboxylic acid reductase enzymes catalyze the ATP and NADPH-dependentreduction of carboxylic acids to their corresponding aldehydes(Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)). TheNocardia iowensis enzyme, encoded by car, was cloned and functionallyexpressed in E. coli (Venkitasubramanian et al., J. Biol. Chem.282:478-485 (2007)). Expression of the npt gene product improvedactivity of the enzyme via post-transcriptional modification. The nptgene encodes a specific phosphopantetheine transferase (PPTase) thatconverts the inactive apo-enzyme to the active holo-enzyme. The naturalsubstrate of this enzyme is vanillic acid, and the enzyme exhibits broadacceptance of aromatic and aliphatic substrates including 4-HB(Venkitasubramanian et al., in Biocatalysis in the Pharmaceutical andBiotechnology Industires, ed. R. N. Patel, Chapter 15, pp. 425-440, CRCPress LLC, Boca Raton, Fla. (2006)).

Gene name GI Number GenBank ID Organism Car 40796035 AAR91681.1 Nocardiaiowensis (sp. NRRL 5646) Npt 114848891 ABI83656.1 Nocardia iowensis (sp.NRRL 5646)

Additional car and npt genes can be identified based on sequencehomology.

Gene name GI Number GenBank ID Organism fadD9 121638475 YP_978699.1Mycobacterium bovis BCG BCG_2812c 121638674 YP_978898.1 Mycobacteriumbovis BCG nfa20150 54023983 YP_118225.1 Nocardia farcinica IFM 10152nfa40540 54026024 YP_120266.1 Nocardia farcinica IFM 10152 SGR_6790182440583 YP_001828302.1 Streptomyces griseus subsp. griseus NBRC 13350SGR_665 182434458 YP_001822177.1 Streptomyces griseus subsp. griseusNBRC 13350 MSMEG_2956 YP_887275.1 YP_887275.1 Mycobacterium smegmatisMC2 155 MSMEG_5739 YP_889972.1 118469671 Mycobacterium smegmatis MC2 155MSMEG_2648 YP_886985.1 118471293 Mycobacterium smegmatis MC2 155MAP1040c NP_959974.1 41407138 Mycobacterium avium subsp.paratuberculosis K-10 MAP2899c NP_961833.1 41408997 Mycobacterium aviumsubsp. paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131Mycobacterium marinum M MMAR_2936 YP_001851230.1 183982939 Mycobacteriummarinum M MMAR_1916 YP_001850220.1 183981929 Mycobacterium marinum MTpauDRAFT_33060 ZP_04027864.1 227980601 Tsukamurella paurometabola DSM20162 TpauDRAFT_20920 ZP_04026660.1 ZP_04026660.1 Tsukamurellapaurometabola DSM 20162 CPCC7001_1320 ZP_05045132.1 254431429 CyanobiumPCC7001 DDBDRAFT_0187729 XP_636931.1 66806417 Dictyostelium discoideumAX4

An additional CAR enzyme found in Streptomyces griseus is encoded by thegriC and griD genes. This enzyme is believed to convert3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde asdeletion of either griC or griD led to accumulation of extracellular3-acetylamino-4-hydroxybenzoic acid, a shunt product of3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot.60(6):380-387 (2007)). Co-expression of griC and griD with SGR_665, anenzyme similar in sequence to the Nocardia iowensis npt, can bebeneficial.

Gene name GI Number GenBank ID Organism griC 182438036 YP_001825755.1Streptomyces griseus subsp. griseus NBRC 13350 Grid 182438037YP_001825756.1 Streptomyces griseus subsp. griseus NBRC 13350

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. This enzyme naturally reduces alpha-aminoadipate toalpha-aminoadipate semialdehyde. The carboxyl group is first activatedthrough the ATP-dependent formation of an adenylate that is then reducedby NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizesmagnesium and requires activation by a PPTase. Enzyme candidates for AARand its corresponding PPTase are found in Saccharomyces cerevisiae(Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al.,Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombeexhibited significant activity when expressed in E. coli (Guo et al.,Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum acceptsS-carboxymethyl-L-cysteine as an alternate substrate, but did not reactwith adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J.Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenumPPTase has not been identified to date

Gene name GI Number GenBank ID Organism LYS2 171867 AAA34747.1Saccharomyces cerevisiae LYS5 1708896 P50113.1 Saccharomyces cerevisiaeLYS2 2853226 AAC02241.1 Candida albicans LYS5 28136195 AAO26020.1Candida albicans Lys1p 13124791 P40976.3 Schizosaccharomyces pombe Lys7p1723561 Q10474.1 Schizosaccharomyces pombe Lys2 3282044 CAA74300.1Penicillium chrysogenum

FIG. 2, Step L—4-Hydroxybutyryl-Phosphate Reductase (EB14)

EB14 catalyzes the reduction of 4-hydroxybutyrylphosphate to4-hydroxybutyraldehyde. An enzyme catalyzing this transformation has notbeen identified to date. However, similar enzymes include phosphatereductases in the EC class 1.2.1. Exemplary phosphonate reductaseenzymes include G3P dehydrogenase (EC 1.2.1.12), aspartate-semialdehydedehydrogenase (EC 1.2.1.11) acetylglutamylphosphate reductase (EC1.2.1.38) and glutamate-5-semialdehyde dehydrogenase (EC 1.2.1.-).Aspartate semialdehyde dehydrogenase (ASD, EC 1.2.1.11) catalyzes theNADPH-dependent reduction of 4-aspartyl phosphate toaspartate-4-semialdehyde. ASD participates in amino acid biosynthesisand recently has been studied as an antimicrobial target (Hadfield etal., Biochemistry 40:14475-14483 (2001)). The E. coli ASD structure hasbeen solved (Hadfield et al., J Mol. Biol. 289:991-1002 (1999)) and theenzyme has been shown to accept the alternate substratebeta-3-methylaspartyl phosphate (Shames et al., J Biol. Chem.259:15331-15339 (1984)). The Haemophilus influenzae enzyme has been thesubject of enzyme engineering studies to alter substrate bindingaffinities at the active site (Blanco et al., Acta Crystallogr. D. Biol.Crystallogr. 60:1388-1395 (2004); Blanco et al., Acta Crystallogr. D.Biol. Crystallogr. 60:1808-1815 (2004)). Other ASD candidates are foundin Mycobacterium tuberculosis (Shafiani et al., J Appl Microbiol98:832-838 (2005)), Methanococcus jannaschii (Faehnle et al., J Mol.Biol. 353:1055-1068 (2005)), and the infectious microorganisms Vibriocholera and Heliobacter pylori (Moore et al., Protein Expr. Purif.25:189-194 (2002)). A related enzyme candidate isacetylglutamylphosphate reductase (EC 1.2.1.38), an enzyme thatnaturally reduces acetylglutamylphosphate toacetylglutamate-5-semialdehyde, found in S. cerevisiae (Pauwels et al.,Eur. J Biochem. 270:1014-1024 (2003)), B. subtilis (O'Reilly et al.,Microbiology 140 (Pt 5):1023-1025 (1994)), E. coli (Parsot et al., Gene.68:275-283 (1988)), and other organisms. Additional phosphate reductaseenzymes of E. coli include glyceraldehyde 3-phosphate dehydrogenase(gapA (Branlant et al., Eur. J. Biochem. 150:61-66 (1985))) andglutamate-5-semialdehyde dehydrogenase (proA (Smith et al., J.Bacteriol. 157:545-551 (1984))). Genes encoding glutamate-5-semialdehydedehydrogenase enzymes from Salmonella typhimurium (Mahan et al., JBacteriol. 156:1249-1262 (1983)) and Campylobacter jejuni (Louie et al.,Mol. Gen. Genet. 240:29-35 (1993)) were cloned and expressed in E. coli.

Protein GenBank ID GI Number Organism asd NP_417891.1 16131307Escherichia coli asd YP_248335.1 68249223 Haemophilus influenzae asdAAB49996 1899206 Mycobacterium tuberculosis VC2036 NP_231670 15642038Vibrio cholera asd YP_002301787.1 210135348 Heliobacter pylori ARG5,6NP_010992.1 6320913 Saccharomyces cerevisiae argC NP_389001.1 16078184Bacillus subtilis argC NP_418393.1 16131796 Escherichia coli gapAP0A9B2.2 71159358 Escherichia coli proA NP_414778.1 16128229 Escherichiacoli proA NP_459319.1 16763704 Salmonella typhimurium proA P53000.29087222 Campylobacter jejuni

FIG. 2, Step M—4-Hydroxybutyryl-CoA Reductase (Alcohol Forming) (EB15)

EB15 enzymes are bifunctional oxidoreductases that convert an4-hydroxybutyryl-CoA to 1,4-butanediol. Enzymes with this activityinclude adhE from E. coli, adhE2 from C. acetobutylicum (Fontaine etal., J. Bacteriol. 184:821-830 (2002)) and the C. acetobutylicum enzymesencoded by bdh I and bdh II (Walter, et al., J. Bacteriol. 174:7149-7158(1992)). In addition to reducing acetyl-CoA to ethanol, the enzymeencoded by adhE in Leuconostoc mesenteroides has been shown to oxide thebranched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya etal., J. Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al., BiotechnolLett, 27:505-510 (2005)).

Protein GenBank ID GI Number Organism adhE NP_415757.1 16129202Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicumbdh I NP_349892.1 15896543 Clostridium acetobutylicum bdh II NP_349891.115896542 Clostridium acetobutylicum adhE AAV66076.1 55818563 Leuconostocmesenteroides adhE NP_781989.1 28211045 Clostridium tetani adhENP_563447.1 18311513 Clostridium perfringens adhE YP_001089483.1126700586 Clostridium difficile

4.3 Example III—Methods of Using Formaldehyde Produced from theOxidation of Methanol in the Formation of Intermediates of CentralMetabolic Pathways for the Formation of Biomass

Provided herein are exemplary pathways, which utilize formaldehydeproduced from the oxidation of methanol (see, e.g., FIG. 1, step J) inthe formation of intermediates of certain central metabolic pathwaysthat can be used for the formation of biomass. Exemplary MMPs forenhancing the availability of reducing equivalents, as well as theproducing formaldehyde from methanol (step J), are provided in FIG. 1.

One exemplary pathway that can utilize formaldehyde produced from theoxidation of methanol (e.g., as provided in FIG. 1) is shown in FIG. 3,which involves condensation of formaldehyde and D-ribulose-5-phosphateto form H6P by EF1 (FIG. 3, step A). The enzyme can use Mg²⁺ or Mn²⁺ formaximal activity, although other metal ions are useful, and evennon-metal-ion-dependent mechanisms are contemplated. H6P is convertedinto F6P by EF2 (FIG. 3, step B).

Another exemplary pathway that involves the detoxification andassimilation of formaldehyde produced from the oxidation of methanol(e.g., as provided in FIG. 1) is shown in FIG. 4 and proceeds throughDHA. EF3 is a special transketolase that first transfers a glycoaldehydegroup from xylulose-5-phosphate to formaldehyde, resulting in theformation of DHA and G3P, which is an intermediate in glycolysis (FIG.4, step A). The DHA obtained from DHA synthase is then furtherphosphorylated to form DHA phosphate by a DHA kinase (FIG. 4, step B).DHAP can be assimilated into glycolysis and several other pathways.

FIG. 3, Steps A and B—Hexulose-6-Phosphate Synthase (EF1) (Step A) and6-phospho-3-hexuloisomerase (EF2) (Step B)

Both of the EF1 and EF2 enzymes are found in several organisms,including methanotrops and methylotrophs where they have been purified(Kato et al., 2006, BioSci Biotechnol Biochem. 70(1):10-21. In addition,these enzymes have been reported in heterotrophs such as Bacillussubtilis also where they are reported to be involved in formaldehydedetoxification (Mitsui et al., 2003, AEM 69(10):6128-32, Yasueda et al.,1999. J Bac 181(23):7154-60. Genes for these two enzymes from themethylotrophic bacterium Mycobacterium gastri MB19 have been fused andE. coli strains harboring the hps-phi construct showed more efficientutilization of formaldehyde (Orita et al., 2007, Appl MicrobiolBiotechnol. 76:439-445). In some organisms, these two enzymes naturallyexist as a fused version that is bifunctional.

Exemplary candidate genes for H6P synthase are:

Protein GenBank ID GI number Organism Hps AAR39392.1 40074227 Bacillusmethanolicus MGA3 Hps EIJ81375.1 387589055 Bacillus methanolicus PB1RmpA BAA83096.1 5706381 Methylomonas aminofaciens RmpA BAA90546.16899861 Mycobacterium gastri YckG BAA08980.1 1805418 Bacillus subtilis

Exemplary gene candidates for EF2 are:

Protein GenBank ID GI number Organism Phi AAR39393.1 40074228 Bacillusmethanolicus MGA3 Phi EIJ81376.1 387589056 Bacillus methanolicus PB1 PhiBAA83098.1 5706383 Methylomonas aminofaciens RmpB BAA90545.1 6899860Mycobacterium gastri

Candidates for enzymes where both of these functions have been fusedinto a single open reading frame include the following.

Protein GenBank ID GI number Organism PH1938 NP_143767.1 14591680Pyrococcus horikoshii OT3 PF0220 NP_577949.1 18976592 Pyrococcusfuriosus TK0475 YP_182888.1 57640410 Thermococcus kodakaraensisNP_127388.1 14521911 Pyrococcus abyssi MCA2738 YP_115138.1 53803128Methylococcus capsulatas

FIG. 4, Step A—Dihydroxyacetone Synthase (EF3)

Another exemplary pathway that involves the detoxification andassimilation of formaldehyde produced from the oxidation of methanol(e.g., as provided in FIG. 1) is shown in FIG. 4 and proceeds throughDHA. EF3 is a special transketolase that first transfers a glycoaldehydegroup from xylulose-5-phosphate to formaldehyde, resulting in theformation of DHA and G3P, which is an intermediate in glycolysis (FIG.4, step A). The DHA obtained from DHA synthase is then furtherphosphorylated to form DHA phosphate by a DHA kinase (FIG. 4, step B).DHAP can be assimilated into glycolysis and several other pathways.

The EF3 enzyme in Candida boidinii uses thiamine pyrophosphate and Mg²⁺as cofactors and is localized in the peroxisome. The enzyme from themethanol-growing carboxydobacterium, Mycobacter sp. strain JC1 DSM 3803,was also found to have DHA synthase and kinase activities (Ro et al.,1997, J Bac 179(19):6041-7). DHA synthase from this organism also hassimilar cofactor requirements as the enzyme from C. boidinii. The K_(m)sfor formaldehyde and xylulose 5-phosphate were reported to be 1.86 mMand 33.3 microM, respectively. Several other mycobacteria, excludingonly Mycobacterium tuberculosis, can use methanol as the sole source ofcarbon and energy and are reported to use EF3 (Part et al., 2003, J Bac185(1):142-7.

Protein GenBank ID GI number Organism DAS1 AAC83349.1 3978466 Candidaboidinii HPODL_2613 EFW95760.1 320581540 Ogataea parapolymorpha DL-1(Hansenula polymorpha DL-1) AAG12171.2 18497328 Mycobacter sp. strainJC1 DSM 3803

FIG. 4, Step B—Dihydroxyacetone (DHA) Kinase

DHA obtained from DHA synthase is further phosphorylated to form DHAphosphate by a DHA kinase. DHAP can be assimilated into glycolysis andseveral other pathways. EF4 has been purified from Ogataea angusta tohomogeneity (Bystrkh, 1983, Biokhimiia, 48(10):1611-6). The enzyme,which phosphorylates DHA and, to a lesser degree, glyceraldehyde, is ahomodimeric protein of 139 kDa. ATP is the preferred phosphate groupdonor for the enzyme. When ITP, GTP, CTP and UTP are used, the activitydrops to about 30%. In several organisms such as Klebsiella pneumoniaeand Citrobacter fruendii (Daniel et al., 1995, JBac 177(15):4392-40),DHA is formed as a result of oxidation of glycerol and is converted intoDHAP by the kinase DHA kinase of K. pneumoniae has been characterized(Jonathan et al, 1984, JBac 160(1):55-60). It is very specific for DHA,with a K_(m) of 4 μM, and has two apparent K_(m) values for ATP, one at25 to 35 μM, and the other at 200 to 300 μM. DHA can also bephosphorylated by glycerol kinases but the DHA kinase from K. puemoniaeis different from glycerol kinase in several respects. While bothenzymes can phosphorylate DHA, DHA kinase does not phosphorylateglycerol, neither is it inhibited by fructose-1,6-diphosphate. InSaccharomyces cerevisiae, DHA kinases (I and II) are involved inrescuing the cells from toxic effects of DHA (Molin et al., 2003, J BiolChem. 17; 278(3):1415-23).

In Escherichia coli, DHA kinase is composed of the three subunits DhaK,DhaL, and DhaM and it functions similarly to a phosphotransferase system(PTS) in that it utilizes phosphoenolpyruvate as a phosphoryl donor(Gutknecht et al., 2001, EMBO J. 20(10):2480-6). It differs in not beinginvolved in transport. The phosphorylation reaction requires thepresence of the EI and HPr proteins of the PTS system. The DhaM subunitis phosphorylated at multiple sites. DhaK contains the substrate bindingsite (Garcia-Alles et al., 2004, 43(41):13037-45; Siebold et al., 2003,PNAS. 100(14):8188-92). The K_(M) for DHA for the E. coli enzyme hasbeen reported to be 6 μM. The K subunit is similar to the N-terminalhalf of ATP-dependent EF4 of Citrobacter freundii and eukaryotes.

Exemplary DHA kinase gene candidates for this step are:

Protein GenBank ID GI number Organism DAK1 P54838.1 1706391Saccharomyces cerevisiae S288c DAK2 P43550.1 1169289 Saccharomycescerevisiae S288c D186_20916 ZP_16280678.1 421847542 Citrobacter freundiiDAK2 ZP_18488498.1 425085405 Klebsiella pneumoniae DAK AAC27705.13171001 Ogataea angusta DhaK NP_415718.6 162135900 Escherichia coli DhaLNP_415717.1 16129162 Escherichia coli DhaM NP_415716.4 226524708Escherichia coli

4.4 Example IV—Methods for Handling Anaerobic Cultures

This example describes methods used in handling anaerobic cultures.

A. Anaerobic chamber and conditions. Exemplary anaerobic chambers areavailable commercially (see, for example, Vacuum Atmospheres Company,Hawthorne Calif.; MBraun, Newburyport Mass.). Conditions included an O₂concentration of 1 ppm or less and 1 atm pure N₂. In one example, 3oxygen scrubbers/catalyst regenerators were used, and the chamberincluded an O₂ electrode (such as Teledyne; City of Industry CA). Nearlyall items and reagents were cycled four times in the airlock of thechamber prior to opening the inner chamber door. Reagents with avolume >5 mL were sparged with pure N₂ prior to introduction into thechamber. Gloves are changed twice/yr and the catalyst containers wereregenerated periodically when the chamber displays increasingly sluggishresponse to changes in oxygen levels. The chamber's pressure wascontrolled through one-way valves activated by solenoids. This featureallowed setting the chamber pressure at a level higher than thesurroundings to allow transfer of very small tubes through the purgevalve.

The anaerobic chambers achieved levels of O₂ that were consistently verylow and were needed for highly oxygen sensitive anaerobic conditions.However, growth and handling of cells does not usually require suchprecautions. In an alternative anaerobic chamber configuration, platinumor palladium can be used as a catalyst that requires some hydrogen gasin the mix. Instead of using solenoid valves, pressure release can becontrolled by a bubbler. Instead of using instrument-based O₂monitoring, test strips can be used instead.

B. Anaerobic microbiology. Serum or media bottles are fitted with thickrubber stoppers and aluminum crimps are employed to seal the bottle.Medium, such as Terrific Broth, is made in a conventional manner anddispensed to an appropriately sized serum bottle. The bottles aresparged with nitrogen for ˜30 min of moderate bubbling. This removesmost of the oxygen from the medium and, after this step, each bottle iscapped with a rubber stopper (such as Bellco 20 mm septum stoppers;Bellco, Vineland, N.J.) and crimp-sealed (Bellco 20 mm). Then thebottles of medium are autoclaved using a slow (liquid) exhaust cycle. Atleast sometimes a needle can be poked through the stopper to provideexhaust during autoclaving; the needle needs to be removed immediatelyupon removal from the autoclave. The sterile medium has the remainingmedium components, for example buffer or antibiotics, added via syringeand needle. Prior to addition of reducing agents, the bottles areequilibrated for 30-60 minutes with nitrogen (or CO depending upon use).A reducing agent such as a 100×150 mM sodium sulfide, 200 mMcysteine-HCl is added. This is made by weighing the sodium sulfide intoa dry beaker and the cysteine into a serum bottle, bringing both intothe anaerobic chamber, dissolving the sodium sulfide into anaerobicwater, then adding this to the cysteine in the serum bottle. The bottleis stoppered immediately as the sodium sulfide solution generateshydrogen sulfide gas upon contact with the cysteine. When injecting intothe culture, a syringe filter is used to sterilize the solution. Othercomponents are added through syringe needles, such as B12 (10 μMcyanocobalamin), nickel chloride (NiCl₂, 20 microM final concentrationfrom a 40 mM stock made in anaerobic water in the chamber and sterilizedby autoclaving or by using a syringe filter upon injection into theculture), and ferrous ammonium sulfate (final concentration needed is100 μM—made as 100-1000× stock solution in anaerobic water in thechamber and sterilized by autoclaving or by using a syringe filter uponinjection into the culture). To facilitate faster growth under anaerobicconditions, the 1 liter bottles were inoculated with 50 mL of apreculture grown anaerobically. Induction of the pAl-lacO1 promoter inthe vectors was performed by addition of isopropylβ-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mMand was carried out for about 3 hrs.

Large cultures can be grown in larger bottles using continuous gasaddition while bubbling. A rubber stopper with a metal bubbler is placedin the bottle after medium addition and sparged with nitrogen for 30minutes or more prior to setting up the rest of the bottle. Each bottleis put together such that a sterile filter will sterilize the gasbubbled in and the hoses on the bottles are compressible with small Cclamps. Medium and cells are stirred with magnetic stir bars. Once allmedium components and cells are added, the bottles are incubated in anincubator in room air but with continuous nitrogen sparging into thebottles.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples and embodiments provided above,it should be understood that various modifications can be made withoutdeparting from the spirit of the invention.

1. A non-naturally occurring microbial organism (NNOMO) having amethanol metabolic pathway (MMP), wherein said organism comprises atleast one exogenous nucleic acid encoding a MMP enzyme (MMPE) expressedin a sufficient amount to enhance the availability of reducingequivalents in the presence of methanol, wherein said MMP comprises: (i)a methanol dehydrogenase (EM9); (ii) an EM9 and a formaldehydeactivating enzyme (EM10); or (iii) a methanol methyltransferase (EM1)and a methylenetetrahydrofolate reductase (EM2).
 2. The organism ofclaim 1, wherein: (a) the MMP comprises: (i) (a) an EM9, amethylenetetrahydrofolate dehydrogenase (EM3), amethenyltetrahydrofolate cyclohydrolase (EM4) and aformyltetrahydrofolate deformylase (EM5); (b) an EM9, an EM3, an EM4 anda formyltetrahydrofolate synthetase (EM6); (c) an EM9 and a formaldehydedehydrogenase (EM11); (d) an EM9, a S-(hydroxymethyl)glutathionesynthase (EM12), a glutathione-dependent formaldehyde dehydrogenase(EM13) and a S-formylglutathione hydrolase (EM14); or (e) an EM9, anEM13 and an EM14; (ii) (a) an EM9, an EM10, an EM3, an EM4 and an EM5;or (b) an EM9, an EM10, an EM3, an EM4 and an EM6; or (iii) (a) an EM1,an EM2, an EM3, an EM4, and an EM5; or (b) an EM1, an EM2, an EM3, anEM4 and an EM6; wherein the MMP optionally further comprises a formatedehydrogenase (EM8), a formate hydrogen lyase (EM15) or a hydrogenase(EM16); and/or wherein said organism optionally comprises two, three,four, five, six or seven exogenous nucleic acids, each encoding a MMPE.3. The organism of claim 1 or 2, further comprising a 1,4-butanediol(BDO) pathway, wherein said organism comprises at least one exogenousnucleic acid encoding a BDO pathway (BDOP) enzyme expressed in asufficient amount to produce BDO, and wherein the BDOP comprises: (i) asuccinyl-CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate(4-HB) dehydrogenase (EB4), a 4-HB kinase (EB5), aphosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase(aldehyde forming) (EB7), and a 1,4-butanediol dehydrogenase (EB8); (ii)an EB3, an EB4, a 4-hydroxybutyryl-CoA transferase (EB11) or a4-hydroxybutyryl-CoA synthetase (EB12), an EB7, and an EB8; (iii) anEB3, an EB4, an EB11 or a 4-hydroxybutyryl-CoA synthetase, and a4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15); (iv) an EB3, anEB4, an EB5, an EB6, and an EB15; (v) an EB3, an EB4, a 4-HB reductase(EB13), and an EB8; (vi) an EB3, an EB4, an EB5, a4-hydroxybutyryl-phosphate reductase (EB14), and an EB8; (vii) asuccinyl-CoA reductase (alcohol forming) (EB10), an EB5, an EB6, an EB7,and an EB8; (viii) an EB10, an EB5, an EB6, and an EB15; (ix) an EB10,an EB11 or an EB12, an EB7, and an EB8; (x) an EB10, an EB11 or an EB12,and an EB15; (xi) an EB10, an EB13, and an EB8; (xii) an EB10, an EB5,an EB14 and an EB8; (xiii) a succinate reductase (EB9), an EB4, an EB5,an EB6, an EB7, and an EB8; (xiv) an EB9, an EB4, an EB11 or an EB12, anEB7, and an EB8; (xv) an EB9, an EB4, an EB11 or an EB12, and an EB15;(xvi) an EB9, an EB4, an EB5, an EB6, and an EB15; (xvii) an EB9, anEB4, an EB13, and an EB8; and (xviii) an EB9, an EB4, an EB5, an EB14,and an EB8; wherein the BDOP optionally further comprises a succinyl-CoAtransferase (EB1) or a succinyl-CoA synthetase (EB2A); and/or whereinthe organism optionally comprises four, five, six or seven exogenousnucleic acids, each encoding a BDOP enzyme.
 4. The organism of claim 1,wherein (a) said microbial organism further comprises one or more genedisruptions, wherein said one or more gene disruptions occur in one ormore endogenous genes encoding protein(s) or enzyme(s) involved innative production of ethanol, glycerol, acetate, lactate, formate, CO₂,and/or amino acids, by said microbial organism, and wherein said one ormore gene disruptions confers increased production of BDO in saidmicrobial organism; and/or (b) wherein one or more endogenous enzymesinvolved in: native production of ethanol, glycerol, acetate, lactate,formate, CO₂ and/or amino acids by said microbial organism, hasattenuated enzyme activity or expression levels.
 5. The organism ofclaim 1, further comprising a formaldehyde assimilation pathway (FAP),wherein said organism comprises at least one exogenous nucleic acidencoding a FAP enzyme (FAPE) expressed in a sufficient amount to producean intermediate of glycolysis and/or a metabolic pathway that can beused in the formation of biomass, and wherein (a) said FAP optionallycomprises a hexulose-6-phosphate synthase (EF1) and a6-phospho-3-hexuloisomerase (EF2); (b) said FAP optionally comprises adihydroxyacetone synthase (EF3) or a dihydroxyacetone kinase (EF4); (c)the intermediate optionally is (i) a hexulose-6-phosphate, afructose-6-phosphate, or a combination thereof; or (ii) adihydroxyacetone, a dihydroxyacetone phosphate, or a combinationthereof; and/or (d) the organism optionally comprises two exogenousnucleic acids, each encoding a FAPE.
 6. The organism of claim 1, wherein(a) said at least one exogenous nucleic acid is a heterologous nucleicacid; (b) said organism is in a substantially anaerobic culture medium;and/or (c) said microbial organism is a species of bacteria, yeast, orfungus.
 7. A method for producing BDO, comprising culturing the organismof claim 3 under conditions and for a sufficient period of time toproduce BDO; wherein said method optionally further comprises separatingthe BDO from other components in the culture, wherein the separationoptionally comprises extraction, continuous liquid-liquid extraction,pervaporation, membrane filtration, membrane separation, reverseosmosis, electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, or ultrafiltration.
 8. Abioderived BDO produced according to the method of claim 7; wherein (a)said bioderived BDO optionally has a carbon-12, carbon-13 and carbon-14isotope ratio that reflects an atmospheric carbon dioxide uptake source;and/or (b) said bioderived BDO optionally has an Fm value of at least80%, at least 85%, at least 90%, at least 95% or at least 98%.
 9. Aculture medium comprising the bioderived BDO of claim 8; wherein (a)said bioderived BDO has a carbon-12, carbon-13 and carbon-14 isotoperatio that reflects an atmospheric carbon dioxide uptake source; and/or(b) said culture medium is separated from the NNOMO having the BDOP. 10.A composition comprising said bioderived BDO of claim 8, and a compoundother than said bioderived BDO; wherein said compound other than saidbioderived BDO optionally is a trace amount of a cellular portion of aNNOMO having a BDOP.
 11. A biobased product comprising the bioderivedBDO of claim 8, wherein said biobased product is (i) a polymer, THF or aTHF derivative, or GBL or a GBL derivative; (ii) a plastic, elasticfiber, polyurethane, polyester, polyhydroxyalkanoate, poly-4-HB,co-polymer of poly-4-HB, poly(tetramethylene ether) glycol,polyurethane-polyurea copolymer, spandex, elastane, Lycra™, or nylon;(iii) a polymer, a resin, a fiber, a bead, a granule, a pellet, a chip,a plastic, a polyester, a thermoplastic polyester, a molded article, aninjection-molded article, an injection-molded part, an automotive part,an extrusion resin, an electrical part and a casing; and optionallywhere the biobased product is reinforced or filled and further where thebiobased product is glass-reinforced or -filled or mineral-reinforced or-filled; (iv) a polymer, wherein the polymer comprises polybutyleneterephthalate (PBT); (v) a polymer, wherein the polymer comprises PBTand the biobased product is a resin, a fiber, a bead, a granule, apellet, a chip, a plastic, a polyester, a thermoplastic polyester, amolded article, an injection-molded article, an injection-molded part,an automotive part, an extrusion resin, an electrical part and a casing;and optionally where the biobased product is reinforced or filled andfurther where the biobased product is glass-reinforced or -filled ormineral-reinforced or -filled; (vi) a THF or a THF derivative, whereinthe THF derivative is polytetramethylene ether glycol (PTMEG), apolyester ether (COPE) or a thermoplastic polyurethane; (viii) a THFderivative, wherein the THF derivative comprises a fiber; or (ix) a GBLor a GBL derivative, wherein the GBL derivative is a pyrrolidone;wherein said biobased product optionally comprises at least 5%, at least10%, at least 20%, at least 30%, at least 40% or at least 50% bioderivedBDO; and/or wherein said biobased product optionally comprises a portionof said bioderived BDO as a repeating unit.
 12. A molded productobtained by molding the biobased product of claim
 10. 13. A process forproducing the biobased product of claim 10, comprising chemicallyreacting said bioderived BDO with itself or another compound in areaction that produces said biobased product.
 14. A polymer comprisingor obtained by converting the bioderived BDO of claim
 8. 15. A methodfor producing a polymer, comprising chemically of enzymaticallyconverting the bioderived BDO of claim 8 to the polymer.
 16. Acomposition comprising the bioderived BDO of claim 8, or a cell lysateor culture supernatant thereof.
 17. A method of producing formaldehyde,comprising culturing the organism of claim 1 under conditions and for asufficient period of time to produce formaldehyde, and optionallywherein the formaldehyde is consumed to provide a reducing equivalent orto incorporate into BDO or target product.
 18. A method of producing anintermediate of glycolysis and/or an intermediate of a metabolic pathwaythat can be used in the formation of biomass, comprising culturing theorganism of claim 5 under conditions and for a sufficient period of timeto produce the intermediate, and optionally wherein the intermediate isconsumed to provide a reducing equivalent or to incorporate into BDO ortarget product.
 19. The method of claim 17 or 18, wherein the organismis cultured in a medium comprising biomass, glucose, xylose, arabinose,galactose, mannose, fructose, sucrose, starch, glycerol, methanol,carbon dioxide, formate, methane, or any combination thereof as a carbonsource.